RESUMEN
The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome contributes to the development of inflammatory diseases. Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease caused by NLRP3 gene mutations that results in excessive IL-1ß production. We previously identified isoliquiritigenin (ILG), a component of Glycyrrhiza uralensis extracts, as a potent inhibitor of the NLRP3 inflammasome. Here, we aimed to investigate whether ILG inhibits the activation of NLRP3 inflammasome caused by NLRP3 gene mutations. We demonstrated that ILG significantly inhibited NLRP3 inflammasome-mediated lactate dehydrogenase (LDH) release and IL-1ß production in two CAPS model THP-1 cell lines, NLRP3-D303N and NLRP3-L353P, in a dose-dependent manner. Interestingly, the NLRP3 inhibitor MCC950 inhibited LDH release and IL-1ß production in NLRP3-D303N cells, but not in NLRP3-L353P cells. Western blotting and caspase-1 activity assays showed that ILG, as well as caspase inhibitors, including Z-VAD and YVAD, suppressed caspase-1 activation. Notably, ILG prevented cryo-sensitive foci formation of NLRP3 without affecting the levels of intracellular Ca2+. We concluded that ILG effectively prevents the constitutive activation of the inflammasome associated with NLRP3 gene mutations by inhibiting the aggregation of cryo-sensitive mutated NLRP3.
Asunto(s)
Caspasa 1 , Chalconas , Síndromes Periódicos Asociados a Criopirina , Inflamasomas , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Chalconas/farmacología , Humanos , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos , Caspasa 1/metabolismo , Caspasa 1/genética , Células THP-1 , Síndromes Periódicos Asociados a Criopirina/tratamiento farmacológico , Síndromes Periódicos Asociados a Criopirina/metabolismo , Síndromes Periódicos Asociados a Criopirina/genética , Interleucina-1beta/metabolismoRESUMEN
Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation.
Asunto(s)
Inmunidad Innata , Interleucina-7 , Ratones , Animales , Células Endoteliales/metabolismo , Papaína , Linfocitos , Pulmón , Inmunidad Adaptativa , Inflamación , Citocinas/metabolismo , Interleucina-33RESUMEN
The potential roles of the gut microbiota in the pathogenesis of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), have attracted increased interest. We have investigated the links between gut microbiota and NASH development in Tsumura-Suzuki non-obese mice fed a high-fat/cholesterol/cholate-based (iHFC) diet that exhibit advanced liver fibrosis using antibiotic treatments. The administration of vancomycin, which targets Gram-positive organisms, exacerbated the progression of liver damage, steatohepatitis, and fibrosis in iHFC-fed mice, but not in mice fed a normal diet. F4/80+-recruited macrophages were more abundant in the liver of vancomycin-treated iHFC-fed mice. The infiltration of CD11c+-recruited macrophages into the liver, forming hepatic crown-like structures, was enhanced by vancomycin treatment. The co-localization of this macrophage subset with collagen was greatly augmented in the liver of vancomycin-treated iHFC-fed mice. These changes were rarely seen with the administration of metronidazole, which targets anaerobic organisms, in iHFC-fed mice. Finally, the vancomycin treatment dramatically modulated the level and composition of bile acid in iHFC-fed mice. Thus, our data demonstrate that changes in inflammation and fibrosis in the liver by the iHFC diet can be modified by antibiotic-induced changes in gut microbiota and shed light on their roles in the pathogenesis of advanced liver fibrosis.
Asunto(s)
Antibacterianos , Ácidos y Sales Biliares , Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Vancomicina , Animales , Ratones , Antibacterianos/farmacología , Ácidos y Sales Biliares/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Vancomicina/farmacologíaRESUMEN
The mammalian immune system consists of two arms, innate and acquired immunity. When I was nominated in 2003 as editorial board members of B.B.R.C., it was in line with the paradigm shift in trends of immunology in the way of thinking from acquired immunity represented by peptide recognition by T-cell receptor to innate immunity represented by the recognition of pathogen-derived molecular pattern including lipid and carbohydrate. In this short perspective, I will introduce hot-spot of recent researches of inflammation, particularly inflammasome and immunometabolism. Here, particular attention is given to small molecule inhibitors of NLRP3 inflammasome activation.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Inmunidad Innata , Inflamación , MamíferosRESUMEN
Transforming growth factor (TGF)-ß1 and prostaglandin E2 (PGE2) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, including melanoma. In this study, we identified a natural compound that attenuated TGF-ß1- and PGE2-induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-ß1- or PGE2-induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-ß1- and PGE2-induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experiments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-ß1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.
Asunto(s)
Dinoprostona , Melanoma Experimental , Factor de Crecimiento Transformador beta1 , Triterpenos , Animales , Dinoprostona/metabolismo , Células Asesinas Naturales , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Ratones , Factor de Crecimiento Transformador beta1/metabolismo , Triterpenos/farmacología , Microambiente TumoralRESUMEN
Osmium is defined in the international council for harmonization (ICH-Q3D) guidelines as an element whose concentration can be determined by validated methods including microwave-assisted nitric acid digestion and inductively coupled plasma mass spectrometry. However, microwave digestion using nitric acid is known to result in osmium recoveries higher than the theoretical values in spiked tests because of the formation of highly volatile osmium tetroxide in an oxidation reaction. To stabilize osmium, the addition of thiourea as a complexing agent has been tested and proved its utility. It remains unclear whether other compounds can prevent the over-recovery of osmium. In this study, we investigated four compounds, thiourea, ascorbic acid, sodium sulfite, and potassium metabisulfite, that could reduce the overestimation of osmium isotopes. The minimum amounts of thiourea, ascorbic acid, sodium sulfite, and potassium metabisulfite required to stabilize 10 ng/mL osmium in blank matrix were 1.0, 1.0, 2.5, and 2.5 g/L, respectively. The relative standard deviations obtained from 12 analyses for each stabilization solution were less than 3.3% in thiourea, 12.7% in ascorbic acid, 9.0% in sodium sulfite, and 10.6% in potassium metabisulfite. The stabilization solutions were investigated in a digested tablet matrix and were found to be effective. The impact of adding stabilization solutions on the determination of all ICH-Q3D element concentrations was also evaluated. As stabilization solutions had a small or significant impact on the determination of some elements, it was concluded that osmium determination should be conducted independently.
Asunto(s)
Microondas , Osmio/análisis , Concentración de Iones de Hidrógeno , Isótopos , Espectrometría de MasasRESUMEN
Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.
Asunto(s)
Macrófagos , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Antígeno CD11c , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Fibrosis , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Macrófagos/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
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.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Inflamación/metabolismo , Neutrófilos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Inflamasomas/metabolismo , Lipólisis/fisiología , Macrófagos/metabolismo , Ratones Transgénicos , Obesidad/metabolismoRESUMEN
The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Antígeno 96 de los Linfocitos/agonistas , Macrófagos/efectos de los fármacos , Modelos Inmunológicos , Receptor Toll-Like 4/agonistas , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular , Células Cultivadas , Biología Computacional , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Diseño de Fármacos , Humanos , Ligandos , Antígeno 96 de los Linfocitos/química , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación del Acoplamiento Molecular , Fosforilación , Piridonas/química , Piridonas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Organismos Libres de Patógenos Específicos , Relación Estructura-Actividad , Receptor Toll-Like 4/química , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
T helper 2 cells produce a number of cytokines including inteleukin (IL)-5, IL-4 and IL-13. Group 2 innate lymphoid cells (ILC2s) also produce IL-5 under sterile conditions. IL-5 is interdigitating homodimeric glycoprotein and a member of the four α helical bundle motifs conserved among hematopoietic cytokines. IL-5 exerts its effects on target cells via IL-5 receptor (IL-5R), composed of an IL-5R α and ßc subunit. The membrane proximal proline-rich motif of the cytoplasmic domain of both IL-5R α and ßc subunits is essential for IL-5 signal transduction. Although IL-5 was initially identified by its ability to support the growth and terminal differentiation of mouse B cells into antibody-secreting cells, recombinant IL-5 exerts pleiotropic activities on various target cells. For example, IL-5 is now recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Overexpression of IL-5 in mouse significantly increases eosinophil numbers and antibody levels in vivo, while mice lacking a functional gene for IL-5 or IL-5R display developmental and functional impairments in B cell and eosinophil lineages. In mice, the role of the IL-5/IL-5R system in the production and secretion of Immunoglobulin (Ig) M and IgA in mucosal tissues has been reported. Although eosinophils protect against invading pathogens including virus, bacteria and helminthes, they are also involved in the pathogenesis of various diseases, such as food allergy, asthma, and inflammatory bowel diseases. The recent expansion in our understanding in the context of IL-5 and IL-5-producing ILC2s in eosinophil activation and the pathogenesis of eosinophil-dependent inflammatory diseases has led to advances in therapeutic options. A new therapy currently under invetigarion in clinical trials uses humanized monoclonal antibodies against IL-5 or the IL-5R. In this review, we summarize our current understanding of the functions of IL-5 and its receptor, the innate regulation of IL-5-producing cells, and therapeutic potential of anti-IL-5 and anti-eosinophil (IL-5R) antibodies.
Asunto(s)
Hipersensibilidad , Interleucina-5/inmunología , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Ensayos Clínicos como Asunto , Eosinófilos/inmunología , Humanos , Hipersensibilidad/tratamiento farmacológico , Hipersensibilidad/inmunología , Interleucina-5/antagonistas & inhibidores , Interleucina-5/genética , Linfocitos/inmunología , Ratones , Receptores de Interleucina-5/inmunología , Receptores de Interleucina-5/metabolismo , Transducción de Señal/inmunología , Células Th2/inmunologíaRESUMEN
Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.
Asunto(s)
Antígenos de Superficie/inmunología , Inflamación/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos CD/inmunología , Antígenos de Superficie/sangre , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Dexametasona/farmacología , Femenino , Masculino , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , Interferón gamma/metabolismo , Riñón/metabolismo , Pulmón/metabolismo , Linfocitos/metabolismo , Animales , Células Cultivadas , Galactosilceramidas/farmacología , Interferón gamma/inmunología , Interferón gamma/farmacología , Interleucina-13/inmunología , Interleucina-13/metabolismo , Interleucina-33/deficiencia , Interleucina-33/genética , Interleucina-5/genética , Interleucina-5/inmunología , Interleucina-5/metabolismo , Riñón/citología , Riñón/efectos de los fármacos , Riñón/inmunología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Fenotipo , Receptores de Interferón/agonistas , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptor de Interferón gammaRESUMEN
Trichophyton infection is highly prevalent and tends to be recurrent. Therefore, it is important to develop new therapeutic agents. Previously, we established a mouse model of Trichophyton-induced contact hypersensitivity (CHS) and demonstrated that dectin-1 was involved in inflammation induced by trichophytin, the Trichophyton antigen. Here, we used that model to investigate glycyrrhetinic acid (GA) from plants of the genus Glycyrrhiza as a potential anti-inflammatory agent against superficial mycoses. GA suppressed swelling and the expression of inflammatory cytokines, including macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA. Anti-MIP-2 antibody suppressed trichophytin-induced inflammation, and antidectin-1 antibody suppressed zymosan-induced MIP-2 production in keratinocyte cells. These results suggest that MIP-2 is produced by dectin-1 activation and is involved in inflammation associated with CHS to trichophytin. GA also suppressed zymosan-induced MIP-2 and interleukin (IL)-8, production in mouse and human macrophages and keratinocytes. Furthermore, GA suppressed the phosphorylation of spleen tyrosine kinase (Syk) and inhibitor of nuclear factor-kappa B (IκBα) and the degradation of IκBα in zymosan-simulated RAW264.7 cells. The results of this study suggest that GA suppresses inflammation induced by trichophytin, partly by the downregulation of Syk phosphorylation.
Asunto(s)
Antiinflamatorios/química , Dermatitis por Contacto/tratamiento farmacológico , Ácido Glicirretínico/química , Lectinas Tipo C/química , Tricofitina/efectos adversos , Animales , Supervivencia Celular , Quimiocina CXCL2/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glycyrrhiza , Inflamación , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Queratinocitos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Micosis/tratamiento farmacológico , Inhibidor NF-kappaB alfa/metabolismo , Fosforilación , Quinasa Syk/metabolismo , Trichophyton , Factor de Necrosis Tumoral alfa/metabolismo , Zimosan/químicaRESUMEN
Dendritic cells (DCs) are proficient APCs that play crucial roles in the immune responses to various Ags and pathogens and polarize Th cell immune responses. Lnk/SH2B adaptor protein 3 (Sh2b3) is an intracellular adaptor protein that regulates B lymphopoiesis, megakaryopoiesis, and expansion of hematopoietic stem cells by constraining cytokine signals. Recent genome-wide association studies have revealed a link between polymorphism in this adaptor protein and autoimmune diseases, including type 1 diabetes and celiac disease. We found that Lnk/Sh2b3 was also expressed in DCs and investigated its role in the production and function of DC lineage cells. In Lnk(-/-) mice, DC numbers were increased in the spleen and lymph nodes, and growth responses of bone marrow-derived DCs to GM-CSF were augmented. Mature DCs from Lnk(-/-) mice were hypersensitive and showed enhanced responses to IL-15 and GM-CSF. Compared to normal DCs, Lnk(-/-) DCs had enhanced abilities to support the differentiation of IFN-γ-producing Th1 cells from naive CD4(+) T cells. This was due to their elevated expression of IL-12Rß1 and increased production of IFN-γ. Lnk(-/-) DCs supported the appearance of IFN-γ-producing T cells even under conditions in which normal DCs supported induction of regulatory T cells. These results indicated that Lnk/Sh2b3 plays a regulatory role in the expansion of DCs and might influence inflammatory immune responses in peripheral lymphoid tissues.
Asunto(s)
Células Dendríticas/inmunología , Interferón gamma/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Linfopoyesis/inmunología , Células TH1/inmunología , Proteínas Adaptadoras Transductoras de Señales , Adyuvantes Inmunológicos/farmacología , Animales , Células de la Médula Ósea/citología , Enfermedad Celíaca/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Diabetes Mellitus Tipo 1/inmunología , Estudio de Asociación del Genoma Completo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/inmunología , Inflamación/inmunología , Interleucina-12/farmacología , Interleucina-15/farmacología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Ganglios Linfáticos/citología , Activación de Linfocitos/inmunología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Receptores de Interleucina-12/biosíntesis , Bazo/citología , Linfocitos T Reguladores/inmunología , Trombopoyesis/inmunologíaRESUMEN
NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.
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Anemia Hemolítica/metabolismo , Eritrocitos/metabolismo , Glucólisis , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Adenosina Trifosfato/metabolismo , Anemia Hemolítica/genética , Animales , Western Blotting , Cromatografía Liquida , Citoplasma/enzimología , Eritrocitos/ultraestructura , Redes y Vías Metabólicas/genética , Metabolómica/métodos , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , NAD/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/deficiencia , Nicotinamida-Nucleótido Adenililtransferasa/genética , Esplenomegalia/genética , Esplenomegalia/metabolismo , Análisis de Supervivencia , Espectrometría de Masas en TándemRESUMEN
The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction.
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Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Homeostasis/inmunología , Intestinos/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos T CD8-positivos/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Homeostasis/genética , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Intestinos/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Ratones , Ratones Noqueados , Polimorfismo Genético/inmunologíaRESUMEN
Stimulation of Toll-like receptor 4 (TLR4) induces not only innate but also adaptive immune responses, and has been suggested to exert adjuvant effects. Additional to such positive effects, pre-stimulation of TLR4 induces endotoxin tolerance where animals are unresponsive to subsequent lethal challenges with lipopolysaccharide (LPS). We examined the effects of pre-stimulation of TLR4 using an agonistic anti-TLR4 mAb (UT12) on antibody production in vivo. Pre-injection of UT12 prior to both primary and secondary immunization completely inhibited antigen-specific antibody responses. Cellular analysis revealed that the inhibition was not due to impairment of T-cell activation. Accordingly, T-helper activities in UT12 pre-injected mice were not impaired. In contrast, B-cell priming was defective in UT12 pre-injected mice. The observation that the expression of activation markers such as CD69 and CD86 on B cells was blocked by UT12 pre-injection supports this. Interestingly, UT12 pre-injection only showed inhibitory effects at the primary and not the secondary immunization. These results provide important information concerning the regulatory mechanisms of antibody production, especially in endotoxin-tolerant states.
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Formación de Anticuerpos/inmunología , Antígenos/inmunología , Linfocitos B/inmunología , Receptor Toll-Like 4/agonistas , Adyuvantes Inmunológicos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Presentación de Antígeno/inmunología , Reacciones Antígeno-Anticuerpo , Reactividad Cruzada/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor Toll-Like 4/inmunologíaRESUMEN
IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5-producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5-producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5-producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5-producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.
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Movimiento Celular/inmunología , Eosinófilos/inmunología , Inmunidad Innata , Interleucina-5/biosíntesis , Neoplasias Pulmonares/inmunología , Melanoma Experimental/inmunología , Escape del Tumor/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Eosinófilos/patología , Femenino , Técnicas de Sustitución del Gen , Interleucina-5/fisiología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Masculino , Melanoma Experimental/patología , Melanoma Experimental/prevención & control , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Immunological memory is characterized by heightened immunoglobulin (Ig) G antibody production caused in part by enhanced plasma cell formation conferred by conserved transmembrane and cytoplasmic segments in isotype-switched IgG B cell receptors. We tested the hypothesis that the IgG tail enhances intracellular B cell antigen receptor (BCR) signaling responses to antigen by analyzing B cells from Ig transgenic mice with IgM receptors or chimeric IgMG receptors containing the IgG tail segment. The IgG tail segment enhanced intracellular calcium responses but not tyrosine or extracellular signal-related kinase (ERK) phosphorylation. Biochemical analysis and crosses to CD22-deficient mice established that IgG tail enhancement of calcium and antibody responses, as well as marginal zone B cell formation, was not due to diminished CD22 phosphorylation or inhibitory function. Microarray profiling showed no evidence for enhanced signaling by the IgG tail for calcium/calcineurin, ERK, or nuclear factor kappaB response genes and little evidence for any enhanced gene induction. Instead, almost half of the antigen-induced gene response in IgM B cells was diminished 50-90% by the IgG tail segment. These findings suggest a novel "less-is-more" hypothesis to explain how switching to IgG enhances B cell memory responses, whereby decreased BCR signaling to genes that oppose marginal zone and plasma cell differentiation enhances the formation of these key cell types.
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Secuencia Conservada , Memoria Inmunológica/inmunología , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/química , Receptores de IgG/metabolismo , Transducción de Señal , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Calcio/metabolismo , Diferenciación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción de Señal/inmunología , Activación TranscripcionalRESUMEN
Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4(+) T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4(+) T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4(+) T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4(+) T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.