RESUMO
Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.
Assuntos
Histiocitose , Receptor 7 Toll-Like , Animais , Camundongos , Citocinas/genética , Histiocitose/genética , Mutação/genética , Nucleosídeos , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genéticaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6Clow patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Nefrite Lúpica/etiologia , Nefrite Lúpica/metabolismo , Monócitos/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Regulação da Expressão Gênica , Imunoglobulina G/imunologia , Imuno-Histoquímica , Imunofenotipagem , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/patologia , Camundongos , Monócitos/metabolismoRESUMO
Toll-like receptors (TLRs) impact myeloid cell responsiveness to environmental cues such as pathogen components and metabolites. Although TLR protein expression in monocytes and tissue macrophages is thought to be optimized for microenvironments in each tissue, a comprehensive study has not been reported. We here examined protein expression of endogenous TLRs in tissue-resident myeloid cells. Neutrophils in peripheral blood, spleen, liver and lung expressed TLR2, TLR4 and TLR5 in all tissues. Ly6C+ MHC IIâ classical monocytes mature into Ly6Câ MHC II+ monocyte-derived dendritic cells (moDCs) or Ly6Câ MHC IIâ patrolling monocytes. These subsets were found in all the tissues studied. TLR2 and TLR4 were displayed on all of these subsets, regardless of location. In contrast, expression of endosomal TLRs did vary with tissues and subsets. moDCs expressed TLR9, but much less TLR7. In contrast, TLR7, not TLR3 or TLR9, was highly expressed in classical and patrolling monocytes. Tissue macrophages such as red pulp macrophages in the spleen, Kupffer cells in the liver, microglia in the brain, alveolar macrophages in the lung and adipose tissue macrophages all expressed TLR2, TLR4 and TLR3. TLR7 was also expressed in these tissue macrophages except Kupffer cells in the liver. TLR9 expression in tissue macrophages was much lower or hard to detect. These results suggest that expression of endosomal TLRs in myeloid cells is influenced by their differentiation status and tissue-specific microenvironments.
Assuntos
Endossomos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores Toll-Like/imunologia , Animais , Células Cultivadas , Camundongos , Receptores Toll-Like/genéticaRESUMO
Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.
Assuntos
Citidina Desaminase/metabolismo , Citidina/análogos & derivados , Citidina/metabolismo , Receptor 8 Toll-Like/metabolismo , Citidina/química , Humanos , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Tumorais Cultivadas , Células U937RESUMO
The small GTPase Arl8b localizes primarily to lysosomes and is involved in lysosomal motility and fusion. Here, we show that Arl8b is required for lysosomal degradation of maternal proteins in the visceral yolk sac endoderm (VYSE), an apical cell layer of the visceral yolk sac, of mouse embryos. The VYSE actively takes up maternal materials from uterine fluid and degrades them in lysosomes to provide breakdown products to the embryo. Arl8b gene-trap mice (Arl8b-/- ) displayed decreased early embryo body size. The Arl8b-/- VYSE exhibited defective endocytic trafficking to the lysosome and accumulation of maternal proteins such as albumin and immunoglobulin G in late endocytic organelles. Furthermore, Transthyretin-Cre;Arl8bflox/flox mice in which Arl8b was ablated specifically in the VYSE also showed decreased embryo body size, defects in trafficking to the lysosome and reduction of the free amino acid level in the embryos. Taken together, these results suggest that Arl8b mediates lysosomal degradation of maternal proteins in the VYSE, thereby contributing to mouse embryonic development.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Saco Vitelino/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Endoderma , Feminino , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , ProteóliseRESUMO
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.
Assuntos
Células Dendríticas/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Antígeno 96 de Linfócito/agonistas , Macrófagos/efeitos dos fármacos , Modelos Imunológicos , Receptor 4 Toll-Like/agonistas , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Biologia Computacional , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Fosforilação , Piridonas/química , Piridonas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Organismos Livres de Patógenos Específicos , Relação Estrutura-Atividade , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Sublingual immunotherapy (SLIT) is effective against allergic rhinitis, although a substantial proportion of individuals is refractory. Herein, we describe a predictive modality to reliably identify SLIT non-responders (NRs). We conducted a 2-year clinical study in 193 adult patients with Japanese cedar pollinosis, with biweekly administration of 2000 Japanese allergy units of cedar pollen extract as the maintenance dose. After identifying high-responder (HR) patients with improved severity scores and NR patients with unchanged or exacerbated symptoms, differences in 33 HR and 34 NR patients were evaluated in terms of peripheral blood cellular profiles by flow cytometry and serum factors by ELISA and cytokine bead array, both pre- and post-SLIT. Improved clinical responses were seen in 72% of the treated patients. Pre-therapy IL-12p70 and post-therapy IgG1 serum levels were significantly different between HR and NR patients, although these parameters alone failed to distinguish NR from HR patients. However, the analysis of serum parameters in the pre-therapy samples with the Adaptive Boosting (AdaBoost) algorithm distinguished NR patients with high probability within the training data set. Cluster analysis revealed a positive correlation between serum Th1/Th2 cytokines and other cytokines/chemokines in HR patients after SLIT. Thus, processing of pre-therapy serum parameters with AdaBoost and cluster analysis can be reliably used to develop a prediction method for HR/NR patients.
Assuntos
Alérgenos/uso terapêutico , Antígenos de Plantas/uso terapêutico , Biomarcadores/metabolismo , Rinite Alérgica/terapia , Imunoterapia Sublingual/métodos , Adulto , Algoritmos , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Análise por Conglomerados , Cryptomeria/imunologia , Citocinas/metabolismo , Feminino , Humanos , Imunoglobulina G/sangue , Interleucina-12/metabolismo , Masculino , Pessoa de Meia-Idade , Pólen/imunologia , Rinite Alérgica/diagnóstico , Rinite Alérgica/imunologia , Índice de Gravidade de Doença , Equilíbrio Th1-Th2 , Resultado do TratamentoRESUMO
Toll-like Receptor 9 (TLR9) is an innate immune receptor recognizing microbial DNA. TLR9 is also activated by self-derived DNA, such as mitochondrial DNA, in a variety of inflammatory diseases. We show here that TLR9 activation in vivo is controlled by an anti-TLR9 monoclonal Ab (mAb). A newly established mAb, named NaR9, clearly detects endogenous TLR9 expressed in primary immune cells. The mAb inhibited TLR9-dependent cytokine production in vitro by bone marrow-derived macrophages and conventional dendritic cells. Furthermore, NaR9 treatment rescued mice from fulminant hepatitis caused by administering the TLR9 ligand CpGB and D-(+)-galactosamine. The production of proinflammatory cytokines induced by CpGB and D-(+)-galactosamine was significantly impaired by the mAb. These results suggest that a mAb is a promising tool for therapeutic intervention in TLR9-dependent inflammatory diseases.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Citocinas/imunologia , DNA/imunologia , Substâncias Protetoras/administração & dosagem , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Endossomos/metabolismo , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/imunologia , Lisossomos/metabolismo , Macrófagos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Toll-like receptor 8 (TLR8) senses single-stranded RNA (ssRNA) and initiates innate immune responses. TLR8 requires proteolytic cleavage at the loop region (Z-loop) between leucine-rich repeat (LRR) 14 and LRR15 for its activation. However, the molecular basis of Z-loop processing remains unknown. To elucidate the mechanism of Z-loop processing, we performed biochemical and structural studies of how the Z-loop affects the function of TLR8. TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. Crystallographic analysis revealed that the uncleaved Z-loop located on the ascending lateral face prevents the approach of the dimerization partner by steric hindrance. This autoinhibition mechanism of dimerization by the Z-loop might be occurring in the proteins of the same subfamily, TLR7 and TLR9.
Assuntos
Processamento de Proteína Pós-Traducional , Receptor 8 Toll-Like/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cristalografia por Raios X , Dimerização , Genes Reporter , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , NF-kappa B/metabolismo , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/genéticaRESUMO
Toll-like receptor (TLR) 7and 8 were considered to recognize single-strand RNA (ssRNA) from viruses. Although these receptors also respond to synthetic small chemical ligands, such as CL075 and R848, it remains to be determined whether these receptors sense natural small molecules or not. In the structure of human TLR8 (huTLR8) with ssRNA, there are two ligand-binding sites: one binds a uridine and the other binds an oligoribonucleotide (ORN). This finding demonstrates that huTLR8 recognizes degradation products of ssRNA, suggesting the presence of natural small ligands. We here show that TLR7 works as the sensor for guanosine (G)/2'-deoxyguanosine (dG) in the presence of ORN where ORN strengthens TLR7 interaction with G/dG. In addition, modified nucleosides such as 7-methylguanosine, 8-hydroxyguanosine (8-OHG) and 8-hydroxydeoxyguanosine (8-OHdG) activated TLR7 with ORNs. Importantly, 8-OHdG-a well-known oxidative DNA damage marker with unknown function-induced strong cytokine production comparable to G and dG both in mouse and human immune cells. Although 8-OHdG bound TLR7/ORN with lower affinity than dG did in isothermal titration calorimetry, administered 8-OHdG was metabolically more stable than dG in the serum, indicating that 8-OHdG acts on TLR7 as an endogenous ligand in vivo To address a role of G analogs in the disease state, we also examined macrophages from Unc93b1 (D34A/D34A) mice, which suffer from TLR7-dependent systemic inflammation, and found that Unc93b1 (D34A/D34A) macrophages showed significantly enhanced response to G alone or 8-OHdG with ORN. In conclusion, our results provide evidence that G, dG, 8-OHG and 8-OHdG are novel endogenous ligands for TLR7.
Assuntos
Guanosina , Macrófagos/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Substituição de Aminoácidos , Animais , Guanosina/análogos & derivados , Guanosina/imunologia , Humanos , Ligantes , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Receptor 7 Toll-Like/genéticaRESUMO
Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1(D34A/D34A) mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.
RESUMO
TLR3 senses viral dsRNA in endolysosomes. The TLR3 ectodomain is cleaved by proteases such as cathepsins in endolysosomes. It remains controversial whether the N-terminal fragment of TLR3 ectodomain (TLR3N) is cleaved off or remains associated with the C-terminal TLR3 fragment (TLR3C). In addition to endosomes, TLR3 is reported to be expressed on the surface of human fibroblasts, but not of human monocyte-derived dendritic cells. Less is known about roles of TLR3N and cell surface TLR3 in dsRNA sensing. In this study, we show the cleavage site of the TLR3 ectodomain and cell surface expression of TLR3 on mouse primary immune cells. TLR3C, which started at 343S, was associated with TLR3N. Both TLR3N and TLR3C were required for activation of IFN-ß and NF-κB promoters by dsRNA, demonstrating that dsRNA is sensed by the TLR3N+C complex. Newly established mAbs to mouse TLR3 revealed that cell surface TLR3 was highly expressed on splenic CD8(+) dendritic cells and marginal zone B cells. Cell surface expression of TLR3 on these cells was dependent on the TLR-specific transporter Unc93B1. Although cell surface TLR3 was only weakly expressed on macrophages, TLR3 mAb specifically enhanced TLR3 responses to dsRNA. These results demonstrate that dsRNA is sensed by the TLR3N+C complex and that cell surface TLR3 is a promising target for modulating TLR3 responses.
Assuntos
Linfócitos B/imunologia , Endossomos/metabolismo , Interferon beta/imunologia , NF-kappa B/imunologia , RNA de Cadeia Dupla/metabolismo , Receptor 3 Toll-Like/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Endossomos/imunologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Regulação da Expressão Gênica , Interferon beta/genética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/imunologia , Transdução de Sinais , Baço/citologia , Baço/imunologia , Receptor 3 Toll-Like/genéticaRESUMO
Toll-like receptors (TLRs) recognize a variety of microbial products and induce defense responses. Pathogen sensing by TLRs occurs either on the cell surface or in endolysosomes. TLR-dependent responses are greatly influenced by the site of pathogen sensing. TLR heterodimers TLR1/TLR2 and TLR2/TLR6 recognize tri- or diacylated microbial lipopeptides, respectively. Although TLR1, 2 and 6 are believed to localize on the cell surface of immune cells, little is known about where lipopeptides are signaled. In this study, we established mAbs to TLR1, 2 and 6. TLR1, 2 and 6 were expressed on the surface of B cells, monocytes and dendritic cells in a manner dependent on a TLR-specific chaperone PRAT4A (protein associated with TLR4 A). Cell surface localization of TLR1 or TLR6 was not necessarily required for TLR2 response. Furthermore, a dynamin inhibitor 'Dynasore' abolished the lipopeptide responses by preventing lipopeptide internalization into LAMP-1 and LAMP-2 positive compartments. Our findings suggest that lipopeptides elicit TLR1/2 and TLR2/6 signaling in the endolysosomes, but not on the cell surface.
Assuntos
Endossomos/metabolismo , Lipopeptídeos/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Dinaminas/metabolismo , Endocitose/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Neutrófilos/metabolismo , Multimerização Proteica , Transporte Proteico , Ratos , Baço/citologia , Baço/metabolismo , Receptor 1 Toll-Like/antagonistas & inibidores , Receptor 1 Toll-Like/química , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/química , Receptor 6 Toll-Like/antagonistas & inibidores , Receptor 6 Toll-Like/químicaRESUMO
BACKGROUND: The eosinophil is deeply associated with the pathogenesis of bronchial asthma and other allergic diseases. We recently identified a novel eosinophil-specific cell surface molecule, major facilitator super family domain containing 10 (Mfsd10). A monoclonal antibody (mAb) against Mfsd10 (M2) showed selective binding and neutralizing activities for eosinophils. However, the relative potency of the blockage of Mfsd10 and other eosinophil-specific molecules for the treatment of allergic diseases has not been evaluated. Therefore, in this study, the effects of M2 and an anti-Siglec-F mAb on antigen-immunized and antigen-specific Th2 cell-transferred murine eosinophilic inflammation models were comparatively investigated. METHODS: Ovalbumin (OVA)-specific Th2 cells were differentiated from naïve CD4+ T cells of DO11.10/RAG-2-/- mice in vitro and cytokine producing activity of the Th2 cells was examined. OVA-immunized and Th2 cell-transferred BALB/c mice were treated with M2 or anti-Siglec-F and challenged with OVA. Then the number of inflammatory cells and the concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) were determined. RESULTS: Antigen-specific Th2 cells produced large amounts of IL-4, IL-5 and IL-13 but not IL-17A or IFN-γ. Administration of M2 significantly suppressed antigen-induced lung eosinophil infiltration both in OVA-immunized and Th2 cell-transferred mice. The potency as well as selectivity of M2 for down-regulating eosinophils was quite similar to that of anti-Siglec-F. Both mAbs did not affect antigen-induced IL-5 production in the lungs. CONCLUSIONS: Mfsd10 as well as Siglec-F could be an effective target to treat eosinophil-related disorders including bronchial asthma.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunossupressores/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Eosinofilia Pulmonar/imunologia , Células Th2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Interleucina-5/biossíntese , Ativação Linfocitária/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Eosinofilia Pulmonar/tratamento farmacológico , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/patologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Células Th2/metabolismoRESUMO
Toll-like receptor 9 (TLR9) is an innate immune sensor for microbial DNA that erroneously responds to self DNA in autoimmune disease. To prevent autoimmune responses, Toll-like receptor 9 is excluded from the cell surface and silenced until the N-terminal half of the ectodomain (TLR9N) is cleaved off in the endolysosome. Truncated Toll-like receptor 9 (TLR9C) senses ingested microbial DNA, although the precise role of the truncation remains controversial. Here we show that TLR9 is expressed on the surface of splenic dendritic cells. Following the cleavage of TLR9 in the endolysosome, N-terminal half of the ectodomain remains associated with truncated TLR9, forming the complex TLR9N+C. The TLR9-dependent cytokine production by Tlr9(-/-) dendritic cells is rescued by a combination of TLR9N and TLR9C, but not by TLR9C alone. These results demonstrate that the TLR9N+C complex is a bona fide DNA sensor.
Assuntos
DNA/metabolismo , Receptor Toll-Like 9/química , Receptor Toll-Like 9/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Células da Medula Óssea/citologia , Membrana Celular/metabolismo , Citocinas/biossíntese , DNA/imunologia , Células Dendríticas/metabolismo , Humanos , Proteínas de Repetições Ricas em Leucina , Lisossomos/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas/metabolismo , Transdução de Sinais , Baço/citologia , Relação Estrutura-Atividade , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/imunologia , Transdução GenéticaRESUMO
OBJECTIVES: The present study was designed to clarify the structure of culture and the three components of attitude in a desirable attitude toward dietary management actions in outpatient haemodialysis patients who are in the maintenance phase of treatment. METHODS: The participants in the study included nine patients undergoing chronic maintenance haemodialysis who have received guidance related to diet and had good test results. Ethnography, by means of participant observation and semi-structured interviews, was chosen as the research method. FINDINGS: Desirable attitude of haemodialysis patients in dietary management actions was found to have a chronological progression in one of the components of attitude: propensity of behaviour. Change in behaviour was influenced by affect and cognition. At the base of the structure of attitude lay three factors: valuing cooking with seasonal ingredients and creating special meals for seasonal occasions; family draws near, shows care and gives support; and belief in information perceived to be good for the health, which was influenced by three components of attitude: affect, cognition, and propensity of behaviour, as well as culture. CONCLUSION: Participants continue to value the food culture that they grew up with, which involves their affect towards, and cognition of, dietary management.
Assuntos
Atitude Frente a Saúde/etnologia , Cultura , Dietoterapia/psicologia , Dieta para Diabéticos/etnologia , Diálise Renal , Idoso , Dieta para Diabéticos/psicologia , Emigrantes e Imigrantes/psicologia , Comportamento Alimentar , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Pacientes AmbulatoriaisRESUMO
Natural immunoglobulin M (IgM) is reactive to autoantigens and is believed to be important for autoimmunity. Blood pentameric IgM loaded with antigens forms a large immune complex (IC) that contains various elements, including apoptosis inhibitor of macrophage (AIM). Here we demonstrate that this IgM-AIM association contributes to autoantibody production under obese conditions. In mice fed a high-fat diet, natural IgM increased through B cell TLR4 stimulation. AIM associated with IgM and protected AIM from renal excretion, increasing blood AIM levels along with the obesity-induced IgM augmentation. Meanwhile, the AIM association inhibited IgM binding to the Fcα/µ receptor on splenic follicular dendritic cells, thereby protecting the IgM IC from Fcα/µ receptor-mediated internalization. This supported IgM-dependent autoantigen presentation to B cells, stimulating IgG autoantibody production. Accordingly, in obese AIM-deficient (AIM(-/-)) mice, the increase of multiple IgG autoantibodies observed in obese wild-type mice was abrogated. Thus, the AIM-IgM association plays a critical role in the obesity-associated autoimmune process.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autoanticorpos/metabolismo , Células Dendríticas Foliculares/metabolismo , Imunoglobulina M/metabolismo , Receptores Imunológicos/metabolismo , Animais , Complexo Antígeno-Anticorpo/sangue , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas Foliculares/imunologia , Dieta Hiperlipídica , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina M/química , Imunoglobulina M/imunologia , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Obesidade/patologia , Plasmócitos/metabolismo , Ligação Proteica , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Depuradores , Receptor 4 Toll-Like/metabolismoRESUMO
Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.
Assuntos
Cisteína/química , Dissulfetos/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteólise , RNA/metabolismo , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Linhagem Celular , Células Cultivadas , Células Dendríticas , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologiaRESUMO
Eosinophilic inflammation is the prominent feature of bronchial asthma, though the importance of eosinophils in the pathogenesis of this disease is controversial. We here established monoclonal antibodies against a newly identified cell surface molecule specifically expressed on mouse eosinophils. Eosinophils were highly purified from small intestine lamina propria and thymus as CD11c(+)Gr1(low)F4/80(+)B220(-) cells. Upon comparative microarray analysis for mRNA expressed in eosinophils and other leukocytes, major facilitator super family domain containing 10 (Mfsd10) was identified as a novel eosinophil-specific cell surface molecule. Hybridomas were established from spleen cells of rats immunized with Mfsd10-introduced Ba/F3 cells. One of three monoclonal antibodies against Mfsd10 displayed selective binding activity against eosinophils recovered in bronchoalveolar lavage fluid of ovalbumin-immunized and -challenged mice. Administration of this antibody in vivo induced a significant reduction of eosinophils recruited in the allergic lungs. Anti-Mfsd10 antibody is useful for investigating the pathophysiological roles of eosinophils with its selective binding and neutralizing activity for mouse eosinophils.
Assuntos
Anticorpos Monoclonais/imunologia , Eosinófilos/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Antígenos/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Análise por Conglomerados , Eosinófilos/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , CamundongosRESUMO
AbstractToll-like receptor 5 (TLR5), a sensor for bacterial flagellin, mounts innate and adaptive immune responses, and has been implicated in infectious diseases, colitis and metabolic syndromes. Although TLR5 is believed to belong to cell surface TLRs, cell surface expression has never been verified. Moreover, it has remained unclear which types of immune cells express TLR5 and contribute to flagellin-dependent responses. In this study we established an anti-mouse TLR5 monoclonal antibody and studied the cell surface expression of TLR5 on immune cells. The macrophage cell line J774 expressed endogenous TLR5 on the cell surface and produced IL-6 and G-CSF in response to flagellin. Cell surface expression of TLR5 and flagellin-induced responses were completely abolished by silencing a TLR-specific chaperone protein associated with TLR4 A (PRAT4A), demonstrating that TLR5 is another client of PRAT4A. In the in vivo immune cells, cell surface TLR5 was mainly found on neutrophils and CD11b (hi) Ly6C (hi) classical monocytes in the bone marrow, circulation, spleen and inflammatory lesions. Ly6C (hi) classical monocytes, but not neutrophils, produced cytokines in response to flagellin. Splenic CD8 (-) CD4 (+) conventional dendritic cells and CD11c (hi) CD11b (hi) lamina propria DCs, also clearly expressed cell surface TLR5. Collectively, cell surface expression of TLR5 is dependent on PRAT4A and restricted to neutrophils, classical monocytes and specific DC subsets.