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1.
Genes Cells ; 29(5): 397-416, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38454012

RESUMO

Staphylococcus aureus is a noteworthy pathogen in allergic diseases, as four staphylococcal exotoxins activate mast cells, a significant contributor to inflammation, in an IgE-independent manner. Although the adhesion of mast cells is an essential process for their immune responses, only a small number of exotoxins have been reported to affect the process. Here, we demonstrated that staphylococcal superantigen-like (SSL) 3, previously identified as a toll-like receptor 2 agonist, induced the adhesion of murine bone marrow-derived mast cells to culture substratum. SSL3-induced adhesion was mediated by fibronectin in an Arg-Gly-Asp (RGD) sequence-dependent manner, suggesting the integrins were involved in the process. Additionally, SSL3 was found to bind to an anti-adhesive surface protein CD43. SSL3 induced the adhesion of HEK293 cells expressing exogenous CD43, suggesting that CD43 is the target molecule for adhesion induced by SSL3. Evaluation of SSL3-derived mutants showed that the C-terminal region (253-326), specifically T285 and H307, are necessary to induce adhesion. SSL3 augmented the IL-13 production of mast cells in response to immunocomplex and SSL12. These findings reveal a novel function of SSL3, triggering cell adhesion and enhancing mast cell activation. This study would clarify the correlation between S. aureus and allergic diseases such as atopic dermatitis.


Assuntos
Adesão Celular , Leucossialina , Mastócitos , Staphylococcus aureus , Superantígenos , Animais , Mastócitos/metabolismo , Mastócitos/imunologia , Camundongos , Humanos , Superantígenos/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/imunologia , Células HEK293 , Leucossialina/metabolismo , Proteínas de Bactérias/metabolismo , Interleucina-13/metabolismo , Camundongos Endogâmicos C57BL
2.
Am J Physiol Cell Physiol ; 326(1): C125-C142, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37955123

RESUMO

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.


Assuntos
Caveolina 1 , Receptores Purinérgicos P2X7 , Animais , Camundongos , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2X7/metabolismo
3.
Genes Cells ; 28(3): 226-236, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36637417

RESUMO

Basophils produce interleukins (IL)-4 in response to various stimuli and may contribute to type 2 immune responses to various infections and allergens. We found that resting basophils freshly isolated from mice produce IL-4 in response to IL-3 but not to high-affinity Fc receptor (FcεRI) cross-linking (CL), yet both required the immunoreceptor tyrosine-based activation motif (ITAM) containing adaptor Fc receptor γ-chain (FcRγ), while basophils activated in vitro by IL-3 become responsive to FcεRI CL. Acquisition of responsiveness to FcεRI CL occurred upon infection with Trichinella spiralis or administration of superantigen. Because cultured basophils return to a quiescent state upon starvation with IL-3 with surface FcεRI levels unchanged, this acquisition is reversible and probably reflects intracellular events requiring protein synthesis. Interestingly, similar activation-associated acquisition was observed for responsiveness to other stimuli, including CD200R3 CL, which is known to signal via DAP-12, and the allergen protease papain. This acquisition of responsiveness to FcεRI CL was inhibited by Jak inhibitor. Thus, the IL-3 signal bifurcates downstream of Jak, into two distinct pathway, one leading to IL-4 production and the other to render basophils competent to respond to stimuli dependent on ITAM-containing adaptors DAP12 and FcRγ for IL-4 production.


Assuntos
Basófilos , Interleucina-3 , Camundongos , Animais , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Basófilos/metabolismo , Interleucina-4/metabolismo , Receptores de IgE/metabolismo , Imunoglobulina E/metabolismo
4.
Genes Cells ; 27(9): 559-567, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35801715

RESUMO

Staphylococcal superantigen-like 12 (SSL12) is reported to evoke the degranulation in murine mast cells. The allelic variant of SSL12 in the genome of reference strain NCTC8325 induced the degranulation of murine mast cells, that of MRSA252 strain did not, nevertheless relatively high sequence similarity (82%). To identify responsible amino acid residues of SSL12 for mast cell activation, we created a series of domain swap mutants and amino acid substitution mutants between the active and inactive variants. The mutants that harbored oligonucleotide/oligosaccharide binding (OB)-fold domain of the active variant activated mast cells. The replacement at position 56 (L56F) in the OB-fold domain diminished the mast cell stimulatory activity, and the combinatorial substitutions L56F/K92E, L56F/D95S, and L56F/S100V abolished the stimulatory activities of the mutant that harbored OB-fold domain of the active variant and the intact active variant. These indicate that the responsive elements of SSL12 for mast cell activation are in the OB-fold of SSL12, and L56 would be an essential amino acid residue for the activation of mast cells. The findings would contribute to the understanding of the molecular mechanism of SSL12 for mast cell activation and the development of toxoids preventing allergic inflammations associated with Staphylococcus aureus.


Assuntos
Infecções Estafilocócicas , Superantígenos , Aminoácidos/metabolismo , Animais , Mastócitos/metabolismo , Camundongos , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Superantígenos/genética , Superantígenos/metabolismo
5.
PLoS Biol ; 18(9): e3000813, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991574

RESUMO

Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ácidos Graxos Voláteis/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Infecções por Salmonella/prevenção & controle , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Feminino , Microbioma Gastrointestinal/imunologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Células U937
6.
Biochem Biophys Res Commun ; 632: 107-112, 2022 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-36206594

RESUMO

Basophils are known to produce a large amount of IL-4 in response to stimuli and play a role in the initiation and propagation of type 2 inflammations. S. aureus secretes a series of pore-forming toxins: α-hemolysin, γ-hemolysins, and leukocidins. In this study, we examined the effects of α-hemolysin, γ-hemolysins (HlgAB and HlgCB), and leukocidins (LukAB, LukED, and Panton-Valentine leukocidin) on the function of basophils. All pore-forming toxins except for Panton-Valentine leukocidin bound to murine bone marrow-derived basophils (BMBs). HlgAB and LukED but not other toxins evoked the leakage of lactate dehydrogenase from BMBs at the concentration of 30 µg/ml γ-hemolysins, HlgAB and HlgCB, induced the secretion of IL-4 in BMBs at concentrations above 3.3 µg/ml. LukAB did not induce, and Hla and LukED induced only a small amount of IL-4. HlgBΔstem, the 5 amino acids deletion mutant of HlgB in the stem region, diminished IL-4 secretion by HlgAB and HlgCB in BMBs. These results suggest that the cell damage and the induction of IL-4 in basophils by HlgAB require pore formation. The induction of IL-4 by γ-hemolysins was also observed in fleshly isolated murine basophils. These results demonstrate a novel function of γ-hemolysins, the induction of IL-4 in basophils, in an IgE-independent manner.


Assuntos
Proteínas Hemolisinas , Interleucina-4 , Animais , Camundongos , Aminoácidos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Basófilos/metabolismo , Exotoxinas/farmacologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Imunoglobulina E , Interleucina-4/metabolismo , Lactato Desidrogenases , Leucocidinas/farmacologia , Staphylococcus aureus/metabolismo
7.
Nat Immunol ; 10(2): 214-22, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19098920

RESUMO

The Fc receptor common gamma-chain (FcRgamma) is a widely expressed adaptor bearing an immunoreceptor tyrosine-based activation motif (ITAM) that transduces activation signals from various immunoreceptors. We show here that basophils lacking FcRgamma developed normally and proliferated efficiently in response to interleukin 3 (IL-3) but were very impaired in IL-3-induced production of IL-4 and in supporting T helper type 2 differentiation. Through its transmembrane portion, FcRgamma associated constitutively with the common beta-chain of the IL-3 receptor and signaled by recruiting the kinase Syk. Retrovirus-mediated complementation demonstrated the essential function of the ITAM of FcRgamma in IL-3 signal transduction. Our results identify a previously unknown mechanism whereby FcRgamma functions to 'route' selective cytokine-triggered signals into the ITAM-mediated IL-4 production pathway.


Assuntos
Basófilos/metabolismo , Interleucina-3/metabolismo , Interleucina-4/biossíntese , Receptores de IgG/metabolismo , Transdução de Sinais/imunologia , Animais , Basófilos/citologia , Basófilos/imunologia , Western Blotting , Diferenciação Celular/imunologia , Proliferação de Células , Citometria de Fluxo , Imunoprecipitação , Interleucina-3/imunologia , Interleucina-4/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia , Receptores de Interleucina-3/imunologia , Receptores de Interleucina-3/metabolismo , Quinase Syk , Células Th2/citologia , Células Th2/imunologia , Ativação Transcricional/imunologia
8.
Biochem Biophys Res Commun ; 532(2): 200-204, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-32859377

RESUMO

S. aureus is associated with atopic dermatitis (AD). Several staphylococcal products including cell wall components, protease, and exotoxins, are thought to be involved in allergic inflammation of AD via activating immune cells such as T cells and mast cells. None of the staphylococcal exotoxins has been reported to activate a primary IL-4 inducer, basophils, that are known to produce large amounts of IL-4 in response to allergens as well as IgE-independent stimuli such as mites and helminth proteases. In this study, we investigated the ability of staphylococcal superantigen-like (SSL) family to activate basophils. SSL12, reported its activity to activate mast cells, induced the production of IL-4 in bone marrow derived basophils. SSL12 also evoked the release of IL-4 in freshly isolated murine basophils in bone marrow cells, as the depletion of basophils by basophils-specific antibodies against high-affinity IgE receptor and CD49b diminished the responsiveness of bone marrow cells for SSL12. These results propose the novel immune regulatory activity of SSL12 by inducing IL-4 in basophils, that contributes to the development of allergic inflammation disorders and the immune evasion of the cocci.


Assuntos
Basófilos/metabolismo , Interleucina-4/metabolismo , Staphylococcus aureus/imunologia , Superantígenos/farmacologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Basófilos/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Interações Hospedeiro-Patógeno , Camundongos Endogâmicos C57BL , Staphylococcus aureus/patogenicidade , Superantígenos/genética , Superantígenos/imunologia
9.
FASEB J ; 33(11): 11821-11835, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31355683

RESUMO

Chronic activation of the IL-1ß system in adipose tissue on metabolic disorders is well demonstrated. However, a mechanism for its expression and activation in the tissue has remained unexplored. Here, we demonstrate that IL-1ß transcript was enriched in neutrophils of white adipose tissue (WAT) from lean mice. Mechanistically, the interaction of neutrophils with adipocytes induced IL-1ß expression via NF-κB pathway. Lipolysis of adipocytes accumulated neutrophils prior to macrophages in WAT and produced high levels of IL-1ß via an inflammasome pathway. Leukotriene B4 (LTB4) production in WAT also contributed to neutrophil accumulation. Furthermore, an LTB4-inflammasome axis contributed to the expression of chemotactic molecules involved in high-fat diet-induced macrophage infiltration into WAT. We have identified previously unappreciated roles for neutrophils in the development of adipose tissue inflammation: robust IL-1ß production and infiltration of macrophages to initiate chronic inflammation.-Watanabe, Y., Nagai, Y., Honda, H., Okamoto, N., Yanagibashi, T., Ogasawara, M., Yamamoto, S., Imamura, R., Takasaki, I., Hara, H., Sasahara, M., Arita, M., Hida, S., Taniguchi, S., Suda, T., Takatsu, K. Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Neutrófilos/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Inflamassomos/metabolismo , Lipólise/fisiologia , Macrófagos/metabolismo , Camundongos Transgênicos , Obesidade/metabolismo
10.
J Biol Chem ; 293(17): 6326-6336, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29496994

RESUMO

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-ß expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-ß, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.


Assuntos
Proteínas de Transporte/metabolismo , Neoplasias do Colo/metabolismo , Proteína DEAD-box 58/metabolismo , Quinase I-kappa B/metabolismo , Interferon beta/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interferon beta/genética , Interferon beta/imunologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores Imunológicos , Viroses/genética , Viroses/imunologia , Viroses/metabolismo
11.
Biochem Biophys Res Commun ; 508(1): 263-269, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497774

RESUMO

Staphylococcal α-hemolysin (Hla) is a principal small ß-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, ß-hexosaminidase. However, Hla more than doubled the releases of ß-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130 mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.


Assuntos
Toxinas Bacterianas/farmacologia , Degranulação Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Proteínas Hemolisinas/farmacologia , Ionomicina/farmacologia , Receptores de IgE/metabolismo , Staphylococcus aureus/química , Animais , Toxinas Bacterianas/química , Sobrevivência Celular/efeitos dos fármacos , Proteínas Hemolisinas/química , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
12.
Biochem Biophys Res Commun ; 511(2): 350-355, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30795864

RESUMO

Staphylococcal superantigen-like (SSL) protein is a family of exotoxins that consists of 14 SSLs, and the roles of several SSLs in immune evasion of the cocci have been revealed. However little is known whether they act as immune activators and are involved in inflammatory disorders such as atopic dermatitis. In this study we examined whether SSLs activate mast cells, the key player of local inflammation. SSL12 evoked the release of a granule enzyme ß-hexosaminidase from bone marrow derived mast cells (BMMCs) in the absence of IgE. The release of the granule enzyme caused by SSL12 was not accompanied with the leakage of a cytosolic enzyme lactate dehydrogenase (LDH), unlike staphylococcal δ-toxin that was reported to induce both the release of ß-hexosaminidase and the leakage of LDH from the cells, suggesting that SSL12 evokes the degranulation of mast cells without cell membrane damage. Furthermore SSL12 induced IL-6 and IL-13 in both mRNA and protein levels indicating that SSL12 induces de novo synthesis of the cytokines. Evans blue extravasation was elevated by the intradermal injection of SSL12, suggesting that SSL12 is also able to evoke local inflammation in vivo. These findings indicate the novel mast cell activating activity of SSLs, and SSL12 is likely an important factor in both initiation phase and effector phase of allergic and immune responses.


Assuntos
Mastócitos/microbiologia , Staphylococcus/imunologia , Superantígenos/imunologia , Animais , Degranulação Celular , Células Cultivadas , Citocinas/imunologia , Interações Hospedeiro-Patógeno , Mastócitos/imunologia , Mastócitos/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
13.
Biochem Biophys Res Commun ; 497(2): 713-718, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29462623

RESUMO

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, ß1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4ß9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved ß1-3 but not α4ß9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted ß1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored ß1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the ß1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.


Assuntos
Proteínas de Bactérias/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Linhagem Celular , Humanos , Metaloproteinase 9 da Matriz/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química
14.
Biochem Biophys Res Commun ; 503(2): 544-549, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29800570

RESUMO

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Células Cultivadas , Feminino , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo
15.
Biol Pharm Bull ; 41(6): 877-884, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29863076

RESUMO

The risk of rheumatoid arthritis (RA) is linked to environmental and genetic factors. Cigarette smoking is an established environmental risk factor for the disease that contributes to its development and severity. Previously, we found that cigarette smoke condensate (CSC), both mainstream and sidestream, aggravates collagen type II-induced arthritis (CIA), which was observed following either intraperitoneal inoculation or nasal exposure. In the present study, we aimed to identify the compound in CSC, which aggravates CIA. By sequential fractionation and analysis, extraction with water/ether in different pH values, silica gel column chromatography, TLC, octadecyl silica (ODS) HPLC, GC/MS, and NMR, the active compound was identified as 5-hydroxy-2-methylpyridine (5H2MP). Its isomer 2-hydroxy-3-methylpyridine, but not 3-hydroxy-2-methylpyridine, was also active. 5H2MP was not mutagenic, and did not exhibit aryl hydrocarbon receptor-dependent activity. Our data help clarify the mechanism underlying the pathogenic effects of cigarette smoking on RA.


Assuntos
Artrite Experimental/patologia , Nicotiana , Piridinas/toxicidade , Fumaça , Animais , Linhagem Celular , Fracionamento Químico , Humanos , Hidrocarbonetos Cíclicos/análise , Masculino , Camundongos Endogâmicos DBA , Testes de Mutagenicidade , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Fumaça/efeitos adversos , Fumaça/análise
16.
Tohoku J Exp Med ; 244(2): 133-144, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29459573

RESUMO

Primary lung cancer is the most frequent cause of cancer-related deaths worldwide. Cisplatin has been used as a key drug in the treatment for patients with lung cancer; however, most of the patients failed to respond to cisplatin within several months, and the mechanisms underlying the cisplatin resistance have not been fully elucidated. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a key adaptor protein in the formation of inflammasomes. ASC is also involved in apoptotic signaling. Importantly, ASC expression is decreased in lung cancer and various cancers, but its precise function in tumor progression remains unknown. To explore the hitherto unknown role of ASC in lung cancer, we initially searched for lung cancer cell lines with higher expression levels of ASC using Cancer Cell Line Encyclopedia (CCLE) database, thereby identifying the A549 human non-small cell lung cancer cell line. Accordingly, with retroviral shRNA, the expression of ASC was forced to decrease in A549 cells. Stable ASC-knockdown cells, thus established, showed the increased activities of proliferation, motility, and invasion, compared with control cells. Importantly, ASC-knockdown cells also became resistant to cisplatin, but not to other anti-cancer agents, 5-fluorouracil and paclitaxel. Bcl-2 and phospho-Src levels were increased in ASC-knockdown cells. A Bcl-2 inhibitor, ABT-199, induced an apoptotic response in ASC-knockdown cells, and dasatinib, a Src inhibitor, blocked cell invasiveness. Thus, ASC may be involved in tumor suppression and cell death via Bcl-2 and pSrc. Targeting Bcl-2 and Src in ASC-downregulated populations of lung cancer may improve treatment outcome.


Assuntos
Apoptose , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Células A549 , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Fenótipo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/efeitos dos fármacos , Quinases da Família src/metabolismo
17.
J Hepatol ; 67(4): 780-790, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28554874

RESUMO

BACKGROUND & AIMS: Although obesity is a risk factor for acute liver failure, the pathogenic mechanisms are not yet fully understood. High cholesterol (HC) intake, which often underlies obesity, is suggested to play a role in the mechanism. We aimed to elucidate the effect of a HC diet on acetaminophen-induced acute liver injury, the most frequent cause of acute liver failure in the USA. METHODS: C57BL/6 Toll-like receptor 9 (TLR9) knockout (Tlr9-/-) mice and their Tlr9+/+ littermates were fed an HC diet for fourweeks and then treated with acetaminophen. Liver sinusoidal endothelial cells (LSECs) were isolated from the mice for in vivo and in vitro analyses. RESULTS: The HC diet exacerbated acetaminophen-induced acute liver injury in a TLR9/inflammasome pathway-dependent manner. LSECs played a major role in the cholesterol loading-induced exacerbation. The accumulation of free cholesterol in the endolysosomes in LSECs enhanced TLR9-mediated signaling, thereby exacerbating the pathology of acetaminophen-induced liver injury through the activation of the TLR9/inflammasome pathway. The accumulation of free cholesterol in LSEC endolysosomes induced a dysfunction of the Rab7 membrane trafficking recycling mechanism, thus disrupting the transport of TLR9 from late endosomes to the lysosomes. Consequently, the level of active TLR9 in the late endosomes increased, thereby enhancing TLR9 signaling in LSECs. CONCLUSIONS: HC intake exaggerated acetaminophen-induced acute liver injury via free cholesterol accumulation in LSECs, demonstrating a novel role of free cholesterol as a metabolic factor in TLR9 signal regulation and pathologies of acetaminophen-induced liver injury. Therapeutic approaches may target this pathway. Lay summary: High cholesterol intake exacerbated acetaminophen-induced acute liver injury via the accumulation of free cholesterol in the endolysosomes of liver sinusoidal endothelial cells. This accumulation enhanced Toll-like receptor 9 signaling via impairment of its membrane trafficking mechanism. Thus, free cholesterol accumulation, as an underlying metabolic factor, exacerbated the pathology of acetaminophen-induced liver injury through activation of the TLR9/inflammasome pathway.


Assuntos
Acetaminofen/toxicidade , Colesterol/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos/farmacologia , Transporte Proteico , Transdução de Sinais , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
18.
Biochem Biophys Res Commun ; 485(1): 201-208, 2017 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-28193526

RESUMO

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10ß1-ß3 (23MEMKN ISALK HGKNN LRFKF RGIKI QVL60) bound to immobilized prothrombin, and mutants that contained SSL10ß10-ß12 (174SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/genética
19.
Microbiol Immunol ; 59(11): 684-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26399380

RESUMO

Interleukin-12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL-2 and IFN-γ. We have previously reported greater IL-12 production from macrophages infected with early-shared BCG sub-strains (ex. BCG-Japan, -Sweden) than from those infected with late-shared BCG (ex. BCG-Pasteur and -Connaught) . In this study, we investigated the Th1 cytokine-inducing activity of splenocytes co-cultured with BCG-infected DCs. Early-shared BCG-infected DCs produced IL-12 and TNF-α⋅ Furthermore, when they were co-cultured with purified protein derivative-stimulated DCs, the splenocytes of mice immunized with BCG-Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG-Connaught. In conclusion, early-shared BCG sub-strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.


Assuntos
Citocinas/biossíntese , Células Dendríticas/fisiologia , Interleucina-12/biossíntese , Infecções por Mycobacterium não Tuberculosas/fisiopatologia , Mycobacterium bovis/patogenicidade , Células Th1/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Técnicas de Cocultura , Feminino , Camundongos
20.
J Immunol ; 188(10): 4838-45, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22504642

RESUMO

NK cell receptors (NKRs) such as NK1.1, NKG2D, and Ly49s are expressed on subsets of CD1d-independent memory phenotype CD8(+) and CD4(-)CD8(-) T cells. However, the mechanism for the generation and functions of these NKR(+) T cells remained elusive. In this study, we found that CD1d-independent Ly49(+) T cells were reduced severely in the spleen, bone marrow, and liver, but not thymus, in mice doubly deficient for IFN regulatory factor-2 (IRF-2) and CD1d, in which the overall memory phenotype T cell population was contrastingly enlarged. Because a large fraction of Ly49(+) T cells coexpressed NK1.1 or NKG2D, the reduction of Ly49(+) T cells resulted indirectly in underrepresentation of NK1.1(+) or NKG2D(+) cells. Ly49(+) T cell deficiency was observed in IRF-2(-/-) mice additionally lacking IFN-α/ßR α-chain (IFNAR1) as severely as in IRF-2(-/-) mice, arguing against the involvement of the accelerated IFN-α/ß signals due to IRF-2 deficiency. Rather, mice lacking IFN-α/ßR alone also exhibited relatively milder Ly49(+) T cell reduction, and IL-2 could expand Ly49(+) T cells from IFNAR1(-/-), but not from IRF-2(-/-), spleen cells in vitro. These results together indicated that IRF-2 acted in Ly49(+) T cell development in a manner distinct from that of IFN-α/ß signals. The influence of IRF-2 deficiency on Ly49(+) memory phenotype T cells observed in this study suggested a unique transcriptional program for this T cell population among other NKR(+) T and memory phenotype T cells.


Assuntos
Antígenos CD1d/fisiologia , Diferenciação Celular/imunologia , Fator Regulador 2 de Interferon/fisiologia , Receptores de Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos CD1d/genética , Diferenciação Celular/genética , Memória Imunológica/genética , Memória Imunológica/imunologia , Imunofenotipagem/métodos , Fator Regulador 2 de Interferon/deficiência , Fator Regulador 2 de Interferon/genética , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Camundongos , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/patologia
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