RESUMEN
Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326+ DCs to dLNs. This effect was abolished by blocking adenosine triphosphate (ATP), one of the DAMPs molecules, as well as by inhibition of Gαi signaling by pertussis toxin. Thus, ICD induction by NIR-PIT stimulates Ti-DC migration to dLNs via ATP-P2X7 receptor and Gαi protein-coupled receptor signaling pathways and may augment tumor antigen presentation to induce anti-tumor T cells in dLNs.
Asunto(s)
Inmunoterapia , Receptores Purinérgicos P2X7 , Ratones , Animales , Toxina del Pertussis , Línea Celular Tumoral , Ratones Desnudos , Muerte Celular Inmunogénica , Células Dendríticas , Adenosina Trifosfato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The evolutionary strategy of transferring maternal antibodies via milk profoundly impacts the survival, lifelong health, and wellbeing of all neonates, including a pronounced impact on human breastfeeding success and infant development. While there has been increased recognition that interorgan connectivity influences the quality of a mother's milk, potentially to personalize it for her offspring, the underlying bases for these processes are incompletely resolved. Here, we define an essential role of Peyer's patches (PPs) for the generation of plasma cells that secrete maternal immunoglobulin A (IgA) into milk. Our metagenomic analysis reveals that the presence of certain residential microorganisms in the gastrointestinal (GI) tract, such as Bacteroides acidifaciens and Prevotella buccalis, is indispensable for the programming of maternal IgA synthesis prior to lactational transfer. Our data provide important insights into how the microbiome of the maternal GI environment, specifically through PPs, can be communicated to the next generation via milk.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Mucosa Intestinal/inmunología , Leche Humana/inmunología , Células Plasmáticas/citología , Animales , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A Secretora/inmunología , Ratones , Ganglios Linfáticos Agregados/inmunologíaRESUMEN
Tumor-infiltrating regulatory T cells (Tregs) have been extensively studied as therapeutic targets. However, not all infiltrating T cells exert their functions equally, presumably because of their heterogeneity and substantial turnover in tissues. In this study, we hypothesized that intertissue migration underlies the functional heterogeneity of Tregs. To test this, we applied in vivo photolabeling to examine single-cell diversity of immunosuppressive molecules in mouse Tregs migrating to, remaining in, and emigrating from MC38 tumors. Neuropilin-1 (Nrp1) expression was inversely correlated with that of six other molecules associated with Treg function. Unsupervised clustering analyses revealed that clusters containing Tregs that were retained in tumors expressed high levels of the six functional molecules but not of Nrp1. However, these clusters represented only half of the Tregs migrating to the tumor, suggesting evolving heterogeneity of tumor-infiltrating Tregs. Thus, we propose progressive pathways of Treg activation and migration between tumors and draining lymph nodes.
Asunto(s)
Adenocarcinoma/inmunología , Neoplasias del Colon/inmunología , Factores de Transcripción Forkhead/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Análisis de la Célula Individual/métodos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Humanos , Activación de Linfocitos , Ratones , Ratones Transgénicos , Neoplasias Experimentales , Neuropilina-1/genética , Neuropilina-1/metabolismo , FenotipoRESUMEN
Immunogenic tumor cell death enhances anti-tumor immunity. However, the mechanisms underlying this effect are incompletely understood. We established a system to induce tumor cell death in situ and investigated its effect on dendritic cell (DC) migration and T cell responses using intravital photolabeling in mice expressing KikGR photoconvertible protein. We demonstrate that tumor cell death induces phagocytosis of tumor cells by tumor-infiltrating (Ti)-DCs, and HMGB1-TLR4 and ATP-P2X7 receptor signaling-dependent Ti-DC emigration to draining lymph nodes (dLNs). This led to an increase in anti-tumor CD8+ T cells of memory precursor effector phenotype and secondary tumor growth inhibition in a CD103+ DC-dependent manner. However, combining tumor cell death induction with lipopolysaccharide treatment stimulated Ti-DC maturation and emigration to dLNs but did not improve tumor immunity. Thus, immunogenic tumor cell death enhances tumor immunity by increasing Ti-DC migration to dLNs where they promote anti-tumor T cell responses and tumor growth inhibition.
RESUMEN
Cell migration and cell proliferation are the basic principles that make up a living organism, and both biologically and medically. In order to understand living organism and biological phenomena, it is essential to track the migration, proliferation, and fate of cells in living cells and animals and to clarify the properties and molecular expression of cells. Recent developments in novel fluorescent proteins have made it possible to observe cell migration and proliferation as the cell cycle at the single-cell level in living individuals and tissues. Here, we introduce cell cycle visualization of living cells and animals by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system and in situ cell labeling of cells and tracking cell migration by photoactivatable and photoconvertible proteins. In addition, we will present our established methods as an example of combines above tools with single-cell molecular expression analysis to reveal the fate of migrating cells at single cell level.
Asunto(s)
Proteínas Luminiscentes , Animales , Ciclo Celular , Movimiento Celular , Proliferación Celular , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genéticaRESUMEN
Dendritic cells (DCs) are essential for successful embryo implantation. However, the properties of uterine DCs (uDCs) during the implantation period are not well characterized. In this study, we investigated the dynamic changes in the uDC phenotypes during the period between coitus and implantation. In virgin mice, we evaluated the expressions of CD103 and XCR1, this is the first report to demonstrate uDCs expressing CD103 in XCR1+cDC1s and XCR1+cDC2s. On day 0.5 post coitus (pc), the number of uterine CD11c+CD103-MHC classIIhighCD86high-mature DCs rapidly increased and then decreased to non-pregnancy levels on days 1.5 and 2.5 pc. On day 3.5 pc just before implantation, the number of CD11c+CD103+MHC class IIdimCD86dim-immature DCs increased in the uterus. The increase in mature uDCs on day 1.5 pc was observed in both allogeneic- and syngeneic mating, suggesting that sexual intercourse, or semen, play a role in this process. Meanwhile, the increase in immature uDCs on day 3.5 pc was only observed in allogeneic mating, suggesting that allo-antigens in the semen contribute to this process. Next, to understand the turnover and migration of uDCs, we monitored DC movement in the uterus and uterine draining lymph nodes (dLNs) using photoconvertible protein Kikume Green Red (KikGR) mice. On day 0.5 pc, uDCs were composed of equal numbers of remaining DCs and migratory DCs. However, on day 3.5 pc, uDCs were primarily composed of migratory DCs, suggesting that most of the uDCs migrate from the periphery just before implantation. Finally, we studied the expression of PD-L2-which induces immunoregulation-on DCs. On day 3.5 pc, PD-L2 was expressed on CD103+-mature and CD103--mature DCs in the uterus. However, PD-L2 expression on CD103--immature DCs and CD103+-immature DCs was very low. Furthermore, both remaining and migratory DCs in the uterus and uterus-derived-DCs in the dLNs on day 3.5 pc highly expressed PD-L2 on their surface. Therefore, our study findings provide a better understanding of the dynamic changes occurring in uterine DCs and dLNs in preparation for implantation following allogeneic- and syngeneic mating.
Asunto(s)
Coito/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Fenotipo , Útero/fisiología , Animales , Biomarcadores , Diferenciación Celular/inmunología , Plasticidad de la Célula/inmunología , Implantación del Embrión/genética , Implantación del Embrión/inmunología , Femenino , Inmunofenotipificación , RatonesRESUMEN
Immune cells are present in the breast milk of several mammalian species; however, their immunological function and transmigration mechanisms to milk remain unknown. Some researchers hypothesize that milk leukocytes have a mammary gland (MG) origin and transmigrate thorough the paracellular pathway, but mammary alveolar epithelial cells strictly regulate the paracellular movement of milk components during lactation via barrier structures, such as tight junctions (TJs). To investigate this discrepancy, we compared leukocyte populations in mouse MG and milk and explored TJ protein expression profiles in MG leukocytes. The main subsets of milk leukocytes were CD8+ and CD4+ T cells displaying the memory phenotype. The proportions of myeloid, B, and dendritic cells were significantly lower in milk than in the MG. CD8+ T cells expressed genes encoding the TJ proteins claudin-3, -7, -12, and ZO-1 at higher levels when compared with myeloid and B cells in the MG among lactating mice. Alveolar epithelial cells in the MG expressed claudin-3, -4, and -7. Administration of FTY720, an inhibitory agonist of sphingosine 1-phosphate receptor 1 that stabilizes TJ permeability, increased the myeloid cell proportion in milk. Different leukocyte populations in the MG and milk suggest active and selective mechanisms of cell transmigration to milk. Both TJ-forming components in alveolar epithelial cells from the MG and TJ protein expression profiles in leukocytes from the MG appear to regulate milk leukocyte populations. T cells are the main population in mouse breast milk and express similar profiles of TJ proteins as those in mammary alveolar epithelial cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/citología , Linfocitos T/inmunología , Proteínas de Uniones Estrechas/metabolismo , Animales , Femenino , Clorhidrato de Fingolimod/administración & dosificación , Factores de Transcripción Forkhead/genética , Humanos , Lactancia , Ratones , Ratones Noqueados , Leche , Embarazo , Proteínas de Uniones Estrechas/genéticaRESUMEN
Regulatory T cells (Tregs) migrate between lymphoid and peripheral tissues for maintaining immune homeostasis. Tissue-specific function and functional heterogeneity of Tregs have been suggested, however, correlation between them and inter-tissue movement remain unknown. We used a contact hypersensitivity model of mice expressing a photoconvertible protein for tracking migratory cells. After marking cells in skin, we purified Tregs exhibiting a different migration pattern [Tregs recruiting to or remaining in the skin and emigrating from the skin to draining lymph nodes (dLNs) within half a day] and examined single-cell gene and protein expression profiles. Correlation and unsupervised clustering analyses revealed that Tregs in both skin and dLNs comprised two subpopulations, one highly expressing Nrp1 with variable CD25, Granzyme B, and/or CTLA-4 expression and another with 3 subsets strongly expressing CD25, Granzyme B, or CTLA-4 together with CD39. Characteristic subsets of Tregs remaining in the skin displayed higher CD25 and CD39 expression and lower Granzyme B and CTLA-4 expression compared with Tregs migrating to the skin. In addition, CCR5 expression in Tregs in skin was positively and negatively correlated with CD39 and Nrp-1 expression, respectively. To assess the predictive value of these data for immunotherapy, we blocked CCR5 signaling and found modest downregulation of CD39 and modest upregulation of Nrp1 expression in skin Tregs. Our data reveal a high functional diversity of Tregs in skin that is strongly related to trafficking behavior, particularly skin retention. Modulation of tissue-specific trafficking and function is a promising clinical strategy against autoimmune, infectious, and neoplastic diseases. Significance Statement: Regulatory T cells (Tregs) are essential for maintaining immune homeostasis. To reveal tissue-specific immunoinhibitory functions and inter-tissue movement correlation based on Treg functional heterogeneity, we examined single-cell gene and protein expression profiles of Tregs recruited to, remaining in, or emigrating from the contact hypersensitivity-induced inflamed skin. Tregs in skin were composed of several subpopulations; one with high Nrp1 expression and another with 3 subsets strongly expressing CD25, Granzyme B, or CTLA-4 together with CD39. Tregs remaining in skin displayed highCD25, CD39, and CCR5 expression, and CCR5 signaling blockade downregulated CD39. A high Treg functional diversity in skin is strongly related to trafficking behavior. Tissue-specific trafficking and functional modulation are a promising clinical strategy against autoimmune, infectious, and neoplastic diseases.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Inflamación/inmunología , Piel/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Biodiversidad , Antígenos CD2/metabolismo , Antígeno CD52/metabolismo , Antígeno CTLA-4/metabolismo , Movimiento Celular , Células Cultivadas , Análisis por Conglomerados , Dermatitis por Contacto , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Análisis de la Célula IndividualRESUMEN
Tetraspanin membrane protein, epithelial membrane protein 3 (Emp3), is expressed in lymphoid tissues. Herein, we have examined the Emp3 in antigen presenting cell (APC) function in the CD8+ cytotoxic T lymphocytes (CTLs) induction. Emp3-overexpressing RAW264.7 macrophage cell line derived from BALB/c mice reduced anti-C57BL/6 alloreactive CTL induction, while Emp3-knockdown RAW264.7 enhanced it compared with parent RAW267.4. Emp3-overexpressing RAW264.7 inhibited, but Emp3-knockdown RAW264.7 augmented, CD8+ T cell proliferation, interferon-γ secretion, IL-2 consumption, and IL-2Rα expression on CD8+ T cells. The supernatant from co-culture with Emp3-overexpressing RAW264.7 contained higher amount of TNF-α, and TNF- α neutralization significantly restored all these inhibitions and the alloreactive CTL induction. These results suggest that Emp3 in allogeneic APCs possesses the inhibitory function of alloreactive CTL induction by downregulation of IL-2Rα expression CD8+ T cells via an increase in TNF-α production. This demonstrates a novel mechanism for regulating CTL induction by Emp3 in APCs through TNF-α production.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/inmunología , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Th17 cells and the cytokine they produce, interleukin (IL)-17, play an important role in tumor progression in humans and in mice. IL-6 and IL-23 are critical cytokines for the differentiation and propagation of Th17 cells, respectively. Bacterial lipopolysaccharides (LPS) are known to stimulate immune cells to produce such inflammatory cytokines. Contrary to Escherichia coli (E. coli) LPS, LPS from Spirulina has low toxicity and barely induces in vivo production of IL-6 and IL-23 in mice. We examined the antitumor effects of Spirulina LPS compared to E. coli LPS in an MH134 hepatoma model. Administration of Spirulina LPS suppressed tumor growth in C3H/HeN mice, but not in Toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice, by reducing serum levels of IL-17 and IL-23, while increasing interferon (IFN)-γ levels. The antitumor activity and IFN-γ production were mediated by T cells. Moreover, in vitro experiments showed that Spirulina LPS impaired the antigen-presenting function that supports the generation of IL-17-producing cells in a toll-like receptor (TLR)4-dependent manner. Of note, injection of anti-IL-17 antibody in tumor-bearing C3H/HeN mice in the absence of Spirulina LPS markedly suppressed tumor growth and augmented IFN-γ responses. Thus, our results support the notion that IFN-γ and IL-17/IL-23 mutually regulate Th17 and Th1 responses in tumor-bearing hosts, and Spirulina LPS modulates the balance of the IFN-γ-IL-17/IL-23 axis towards IFN-γ production, which leads to tumor inhibition. Furthermore, Spirulina LPS effectively inhibited the spontaneous development of mammary tumors. This study has important implications for the exploitation of TLR-based immunomodulators for cancer immunotherapy.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Interferón gamma/inmunología , Interleucina-17/inmunología , Interleucina-23/inmunología , Lipopolisacáridos/farmacología , Spirulina/química , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/prevención & control , Activación de Linfocitos/efectos de los fármacos , Linfoma de Células T/inmunología , Linfoma de Células T/metabolismo , Linfoma de Células T/prevención & control , Ratones , Ratones Endogámicos C3H , Receptor Toll-Like 4/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TLR2 associates with TLR1 and recognizes microbial lipoproteins. Pam3CSK4, a triacylated lipoprotein, is anchored to the extracellular domain of TLR1 and TLR2 and induces pro-inflammatory signals. Here we show that C4b binding protein (C4BP), which is a complement pathway inhibitor, is a TLR2-associated molecule. Immunoprecipitation assay using anti-TLR2 mAb shows that C4BP binds to TLR2. In C4BP-deficient mice, Pam3CSK4-induced IL-6 levels were increased compared with wild type mice. In C4BP-expressing cells, Pam3CSK4-induced IL-8 production was reduced depending on the C4BP expression levels. These results reveal the important role of C4BP in negative regulation of TLR1/2-dependent pro-inflammatory cytokine production. Furthermore, using a fluorescent conjugated Pam3CSK4, we show that C4BP blocks the binding of Pam3CSK4 to TLR1/2. Finally, we show that exogenous C4BP also inhibits Pam3CSK4-induced signaling leading to IL-8 production. Our results indicate C4BP binding to TLR2 and consequent neutralization of its activity otherwise inducing pro-inflammatory cytokine production. C4BP is a negative regulator of TLR1/2 activity.
Asunto(s)
Proteína de Unión al Complemento C4b/metabolismo , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Sitios de Unión , Activación de Complemento , Células HEK293 , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopéptidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Transducción de SeñalRESUMEN
This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down-regulating angiogenesis via a Toll-like receptor 4 signal. Murine RSV-M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV-M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)-17 in both C3H/HeN and C3H/HeJ tumor-bearing mice. Treatment with E. coli LPS induced much greater IL-17 production in tumor-bearing C3H/HeN mice than in tumor-bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re-transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti-cluster of differentiation (CD)8, anti-CD4, anti-CD8 antibodies, and anti-asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti-interferon-γ antibodies had no effect on glioma cell growth, anti-IL-17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS-treated mice than in those from saline- or E. coli LPS-treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down-regulating angiogenesis, and that this down-regulation is mediated in part by regulating IL-17 production.
Asunto(s)
Antineoplásicos/metabolismo , Glioma/tratamiento farmacológico , Factores Inmunológicos/inmunología , Neovascularización Patológica , Polisacáridos Bacterianos/inmunología , Spirulina/química , Receptor Toll-Like 4/metabolismo , Animales , Antineoplásicos/aislamiento & purificación , Línea Celular Tumoral , Escherichia coli/inmunología , Femenino , Glioma/patología , Factores Inmunológicos/aislamiento & purificación , Interleucina-17/metabolismo , Células Asesinas Naturales/inmunología , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Polisacáridos Bacterianos/aislamiento & purificación , Linfocitos T/inmunología , Receptor Toll-Like 4/deficienciaRESUMEN
The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana Bacteriana Externa/fisiología , Células Epiteliales/microbiología , Gammaproteobacteria/metabolismo , Encía/microbiología , Animales , Células Epiteliales/citología , Escherichia coli/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Modelos Genéticos , Mutación , Fosforilación , Transducción de Señal , Regulación hacia ArribaRESUMEN
Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9.
Asunto(s)
Catepsinas/fisiología , Receptor Toll-Like 9/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Catepsinas/antagonistas & inhibidores , Catepsinas/genética , Línea Celular , Islas de CpG/inmunología , ADN Bacteriano/inmunología , Inhibidores Enzimáticos/farmacología , Técnicas de Transferencia de Gen , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/fisiología , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacocinética , ARN Mensajero/metabolismo , Bazo/citología , Receptor Toll-Like 9/genéticaRESUMEN
LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-alpha, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-kappaB through TLR4/MD-2.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Hepatitis/inmunología , Hepatitis/prevención & control , Antígeno 96 de los Linfocitos/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Enfermedad Aguda , Animales , Apoptosis/inmunología , Línea Celular , Epítopos/inmunología , Galactosamina/farmacología , Hepatitis/metabolismo , Hepatitis/patología , Humanos , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/inmunología , Antígeno 96 de los Linfocitos/metabolismo , Ratones , FN-kappa B/metabolismo , Unión Proteica , Choque Séptico/inducido químicamente , Choque Séptico/inmunología , Choque Séptico/prevención & control , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Toll-like receptors (TLRs) recognize microbial products and induce immune responses. Their subcellular distribution is believed to be optimized for their pathogen recognition. Little is known, however, about molecular mechanisms regulating the subcellular distribution of TLR. Lipopolysaccharide, a principal membrane component of the Gram-negative bacteria, is recognized by the receptor complex consisting of Toll-like receptor 4 (TLR4) and MD-2. We here show that a novel molecule, a PRotein Associated with Tlr4 (PRAT4B), regulates cell surface expression of TLR4. PRAT4B has a signal peptide followed by a mature peptide. PRAT4B is associated with the hypoglycosylated, immature form of TLR4 but not with MD-2 or TLR2. Downregulation of PRAT4B mRNA with small interfering RNA decreased cell surface TLR4 on HEK293 cells. These results suggest a novel mechanism regulating the subcellular distribution of TLR4.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Riñón/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Secuencia Conservada , Regulación de la Expresión Génica/fisiología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
Hyaluronan (HA), is a high molecular mass extracellular matrix constituting connective tissue and plays a critical role in not only homeostasis but also inflammatory and wound-healing responses. In this study, we investigated the effect of fibroblast growth factor (FGF)-2 on the production of HA by human dental pulp cells (HDPC). An inhibition binding-protein assay showed that FGF-2 increased HA production by HDPC. In addition, expression of mRNA of hyaluronan synthase (HAS) 1 and HAS 2, both of which are related to the production of high molecular mass of HA, but not HAS 3, was enhanced in FGF-2-stimulated HDPC. These results provide new evidence for the involvement of FGF-2 in the regulation of HA production by HDPC possibly through HAS 1 and HAS 2.
Asunto(s)
Pulpa Dental/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ácido Hialurónico/análisis , Células Cultivadas , Pulpa Dental/citología , Glucuronosiltransferasa/análisis , Glucuronosiltransferasa/efectos de los fármacos , Humanos , Hialuronano Sintasas , Isoenzimas/análisis , Isoenzimas/efectos de los fármacos , ARN Mensajero/análisisRESUMEN
Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105(-/-) B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105(-/-) mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105(-/-) spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was approximately 3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Antígenos CD/fisiología , Antígenos Ly/metabolismo , Antígenos de Superficie/fisiología , Escherichia coli/inmunología , Glicoproteínas de Membrana/fisiología , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Antibacterianos/sangre , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación/genética , Antígenos Ly/genética , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Membrana Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ligandos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 9RESUMEN
Toll-like receptor 4 (TLR4) and MD-2 recognizes lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4-MD-2. Here we show ligand-induced TLR4 oligomerization, homotypic interaction of TLR4, which directly leads to TLR4 signaling. Since TLR4 oligomerization normally occurred in the absence of the cytoplasmic portion of TLR4, TLR4 oligomerization works upstream of TLR4 signaling. Lipid IVa, a lipid A precursor, is agonistic on mouse TLR4-MD-2 but turns antagonistic on chimeric mouse TLR4-human MD-2, demonstrating that the antagonistic activity of lipid IVa is determined by human MD-2. Binding studies with radioactive lipid A and lipid IVa revealed that lipid IVa is similar to lipid A in dose-dependent and saturable binding to mouse TLR4-human MD-2. Lipid IVa, however, did not induce TLR4 oligomerization, and inhibited lipid A-dependent oligomerization of mouse TLR4-human MD-2. Thus, lipid IVa binds mouse TLR4-human MD-2 but does not trigger TLR4 oligomerization. Binding study further revealed that the antagonistic activity of lipid IVa correlates with augmented maximal binding to mouse TLR4-human MD-2, which was approximately 2-fold higher than lipid A. Taken together, lipid A antagonist lipid IVa is distinct from lipid A in binding to TLR4-MD-2 and in subsequent triggering of TLR4 oligomerization. Given that the antagonistic activity of lipid IVa is determined by MD-2, MD-2 has an important role in a link between ligand interaction and TLR4 oligomerization.
Asunto(s)
Antígenos Ly/inmunología , Antígenos de Superficie/inmunología , Proteínas Portadoras/inmunología , Glucolípidos/inmunología , Lípido A/análogos & derivados , Lípido A/antagonistas & inhibidores , Lípido A/inmunología , Complejos Multiproteicos/inmunología , Receptores de Superficie Celular/inmunología , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Glucolípidos/metabolismo , Humanos , Lípido A/metabolismo , Antígeno 96 de los Linfocitos , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 4RESUMEN
BACKGROUND: The mechanism of stimulation of human gingival epithelial cells (HGEC) by Porphyromonas gingivalis (Pg) has not been fully clarified yet. In order to investigate the possible activation of HGEC by Pg through Toll-like receptors (TLRs), we analyzed the production of chemotactic factors and the activated nuclear factor-kappa B (NF-kappaB). METHODS: The mRNA expression of TLRs and the protein expression of TLR2 and TLR4 in HGEC and gingival tissue were assessed using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunohistochemical staining. Primary cultured HGEC (nHGEC) and HGEC transformed by simian virus 40 T antigen (OBA-9) were activated by a sonic extract (SE) of Pg to examine cytokine production and NF-kappaB activation using enzyme-linked immunosorbant assay (ELISA). In addition, Pg mediated activation of NF-kappaB in a TLR2-transfectant was also investigated. RESULTS: RT-PCR results revealed that HGEC expressed mRNA of TLR2, TLR4, TLR5, and TLR9, although the expression profiles of each cell line were slightly different. In addition, immunostaining revealed the prominent expression of TLR2 not only in nHGEC, but also in the gingival epithelium of the tissue specimen. Interestingly, nHGEC and OBA-9 secreted IL-8 and monocyte chemoattractant protein (MCP)-1 upon stimulation with Pg SE more efficiently than LPS and fimbriae of Pg. Furthermore, Pg SE increased the activated NF-kappaB not only in OBA-9, but also in 293T cells transfected with the human TLR2 gene. CONCLUSION: TLR2 participates, at least partly, in the signaling pathway to induce chemokine production in gingival epithelium as a reaction against Pg component(s), probably other than lipopolysaccharide and fimbriae.