RESUMEN
In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.
Asunto(s)
Astrocitos/patología , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Gliosis/patología , Neuroprotección/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Animales , Encéfalo/citología , Encéfalo/inmunología , Movimiento Celular , Proliferación Celular , Quimiocina CCL1/inmunología , Quimiocina CCL20/inmunología , Interleucina-2/inmunología , Interleucina-33/inmunología , Interleucina-6/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Receptores CCR/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Factor de Transcripción STAT3/metabolismo , Serotonina/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismoRESUMEN
Lymphocytes such as CD4+ T cells and B cells mainly infiltrate the salivary glands; however, the precise roles and targets of autoreactive T cells and autoantibodies in the pathogenesis of Sjögren's Syndrome (SS) remain unclear. This study was designed to clarify the role of autoreactive T cells and autoantibodies at the single-cell level involved in the development of sialadenitis. Infiltrated CD4+ T and B cells in the salivary glands of a mouse model resembling SS were single-cell-sorted, and their T cell receptor (TCR) and B cell receptor (BCR) sequences were analyzed. The predominant TCR and BCR clonotypes were reconstituted in vitro, and their pathogenicity was evaluated by transferring reconstituted TCR-expressing CD4+ T cells into Rag2-/- mice and administering recombinant IgG in vivo. The reconstitution of Th17 cells expressing TCR (#G) in Rag2-/- mice resulted in the infiltration of T cells into the salivary glands and development of sialadenitis, while an autoantibody (IgGr22) was observed to promote the proliferation of pathogenic T cells. IgGr22 specifically recognizes double-stranded RNA (dsRNA) and induces the activation of dendritic cells, thereby enhancing the expression of IFN signature and inflammatory genes. TCR#G recognizes antigens related to the gut microbiota. Antibiotic treatment severely reduces the activation of TCR#G-expressing Th17 cells and suppresses sialadenitis development. These data suggest that the anti-dsRNA antibodies and, TCR recognizing the gut microbiota involved in the development of sialadenitis like SS. Thus, our model provides a novel strategy for defining the roles of autoreactive TCR and autoantibodies in the development and pathogenesis of SS.
Asunto(s)
Autoanticuerpos , Receptores de Antígenos de Linfocitos T , Sialadenitis , Síndrome de Sjögren , Animales , Síndrome de Sjögren/inmunología , Sialadenitis/inmunología , Autoanticuerpos/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Ratones Noqueados , Glándulas Salivales/inmunología , Ratones Endogámicos C57BL , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Linfocitos B/inmunología , Células Th17/inmunología , Femenino , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/genéticaRESUMEN
T cells play an important role in the acquired immune response, with regulatory T cells (Tregs) serving as key players in immune tolerance. Tregs are found in nonlymphoid and damaged tissues and are referred to as "tissue Tregs". They have tissue-specific characteristics and contribute to immunomodulation, homeostasis, and tissue repair through interactions with tissue cells. However, important determinants of Treg tissue specificity, such as antigen specificity, tissue environment, and pathology, remain unclear. In this study, we analyzed Tregs in the central nervous system of mice with ischemic stroke and experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. The gene expression pattern of brain Tregs in the EAE model was more similar to that of ischemic stroke Tregs in the brain than to that of spinal cord Tregs. In addition, most T-cell receptors (TCRs) with high clonality were present in both the brain and spinal cord. Furthermore, Gata3+ and Rorc+ Tregs expressed TCRs recognizing MOG in the spinal cord, suggesting a tissue environment conducive to Rorc expression. Tissue-specific chemokine/chemokine receptor interactions in the spinal cord and brain influenced Treg localization. Finally, spinal cord- or brain-derived Tregs had greater anti-inflammatory capacities in EAE mice, respectively. Taken together, these findings suggest that the tissue environment, rather than pathogenesis or antigen specificity, is the primary determinant of the tissue-specific properties of Tregs. These findings may contribute to the development of novel therapies to suppress inflammation through tissue-specific Treg regulation.
Asunto(s)
Encéfalo , Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Médula Espinal , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Ratones , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Médula Espinal/inmunología , Médula Espinal/patología , Médula Espinal/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Modelos Animales de EnfermedadRESUMEN
Cytokines are important intercellular communication tools for immunity. Many cytokines promote gene transcription and proliferation through the JAK/STAT (Janus kinase/signal transducers and activators of transcription) and the Ras/ERK (GDP/GTP-binding rat sarcoma protein/extracellular signal-regulated kinase) pathways, and these signaling pathways are tightly regulated. The SOCS (suppressor of cytokine signaling) family members are representative negative regulators of JAK/STAT-mediated cytokine signaling and regulate the differentiation and function of T cells, thus being involved in immune tolerance. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergy and tumorigenesis. SOCS family proteins also function as immune-checkpoint molecules that contribute to the unresponsiveness of T cells to cytokines.
Asunto(s)
Citocinas/inmunología , Tolerancia Inmunológica/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Animales , Humanos , Transducción de Señal/inmunologíaRESUMEN
Cytokines are important intercellular communication tools for immunity. Most cytokines utilize the JAK-STAT and Ras-ERK pathways to promote gene transcription and proliferation; however, this signaling is tightly regulated. The suppressor of cytokine signaling (SOCS) family and SPRED family are a representative negative regulators of the JAK-STAT pathway and the Ras-ERK pathway, respectively. The SOCS family regulates the differentiation and function of CD4+ T cells, CD8+ T cells, and regulatory T cells, and is involved in immune tolerance, anergy, and exhaustion. SPRED family proteins have been shown to inactivate Ras by recruiting the Ras-GTPase neurofibromatosis type 1 (NF1) protein. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergies, and tumorigenesis, and SPRED1 is involved in NF1-like syndromes and tumors. We also identified the NR4a family of nuclear receptors as a key transcription factor for immune tolerance that suppresses cytokine expression and induces various immuno-regulatory molecules including SOCS1.
Asunto(s)
Linfocitos T CD8-positivos , Proteínas Supresoras de la Señalización de Citocinas , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Humanos , Tolerancia Inmunológica , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Inflammation and immune responses after tissue injury play pivotal roles in the pathology, resolution of inflammation, tissue recovery, fibrosis and remodeling. Regulatory T cells (Tregs) are the cells responsible for suppressing immune responses and can be activated in secondary lymphatic tissues, where they subsequently regulate effector T cell and dendritic cell activation. Recently, Tregs that reside in non-lymphoid tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Unlike other tissue Tregs, the role of Tregs in the brain has not been well elucidated because the number of brain Tregs is very small under normal conditions. However, we found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis through secretion of a cytokine, amphiregulin (Areg). Brain Tregs resemble other tissue Tregs in many ways but, unlike the other tissue Tregs, brain Tregs express neural-cell-specific genes such as the serotonin receptor (Htr7) and respond to serotonin. Administering serotonin or selective serotonin reuptake inhibitors (SSRIs) in an experimental mouse model of stroke increases the number of brain Tregs and ameliorates neurological symptoms. Knowledge of brain Tregs will contribute to the understanding of various types of neuroinflammation.
Asunto(s)
Astrocitos/inmunología , Encéfalo/inmunología , Inflamación/inmunología , Daño por Reperfusión/inmunología , Linfocitos T Reguladores/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Receptores de Serotonina/metabolismo , Cicatrización de HeridasRESUMEN
Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Dioxigenasas/inmunología , Factores de Transcripción Forkhead/metabolismo , Interleucina-17/biosíntesis , Proteínas Proto-Oncogénicas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Animales , Proliferación Celular , Interleucina-17/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Viable populations of the cheilostome bryozoan Cribrilina mutabilis Ito, Onishi & Dick exist in the NW Pacific (Russian Far East and northern Japan), NE Atlantic (Scandinavia and Scotland), and NW Atlantic (Maine, USA). The first NE and NW Atlantic records are from Norway (2008) and Casco Bay, Maine, USA (2018), respectively, indicating a relatively recent introduction to the region. Mitochondrial COI gene sequences from North Atlantic populations (Sweden, Norway, and Maine) showed two haplotypes differing by one substitution, but differed from two haplotypes from Akkeshi, northern Japan, by 6-8 substitutions. North Atlantic populations differed morphologically from the Akkeshi population in that some zooids formed a suboral projection, and frontal zooids were more common. While C. mutabilis in northern Japan has been found only on natural or artificial eelgrass (Zostera marina), across its range it has been found on several species of algae, plastic panels and strips, several species of Zostera, and mollusc shells. Similar frequencies of heteromorphic zooids with differing degree of frontal wall calcification, i.e., R (rib)-, I (intermediate)-, and S (shield)-type zooids, in colonies on eelgrass at comparable times of the season and across populations suggest an innate response to seasonal environmental fluctuations, although zooid frequencies were different on non-eelgrass substrates. The increase in trans-Arctic shipping along the Northern Sea Route in recent decades, and previous documentation of C. mutabilis on ship hulls in the Sea of Japan, indicate a clear mechanism for anthropogenic introduction from the Far East to Europe in recent decades.
Asunto(s)
Distribución Animal , Briozoos/fisiología , Especies Introducidas , Animales , Briozoos/genética , Complejo IV de Transporte de Electrones/análisis , Genes Mitocondriales , Variación Genética , Haplotipos , Rasgos de la Historia de Vida , Análisis de Secuencia de ADNRESUMEN
T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-ß). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-ß-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.
Asunto(s)
Reprogramación Celular , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Células TH1/citología , Células TH1/metabolismo , Adulto , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología , Células TH1/inmunologíaRESUMEN
Damage-associated molecular patterns (DAMPs) have been implicated in sterile inflammation in various tissue injuries. High-mobility group box 1 (HMGB1) is a representative DAMP, and has been shown to transmit signals through receptors for advanced glycation end products (RAGEs) and TLRs, including TLR2 and TLR4. HMGB1 does not, however, bind to TLRs with high affinity; therefore, the mechanism of HMGB1-mediated TLR activation remains unclear. In this study, we found that fluorescently labeled HMGB1 was efficiently internalized into macrophages through class A scavenger receptors. Although both M1- and M2-type macrophages internalized HMGB1, only M1-type macrophages secreted cytokines in response to HMGB1. The pan-class A scavenger receptor competitive inhibitor, maleylated bovine serum albumin (M-BSA), inhibited HMGB1 internalization and reduced cytokine production from macrophages in response to HMGB1 but not to LPS. The C-terminal acidic domain of HMGB1 is responsible for scavenger receptor-mediated internalization and cytokine production. HMGB1 and TLR4 co-localized in macrophages, and this interaction was disrupted by M-BSA, suggesting that class A scavenger receptors function as co-receptors of HMGB1 for TLR activation. M-BSA ameliorated LPS-induced sepsis and dextran sulfate sodium (DSS)-induced colitis models in which HMGB1 has been shown to play progressive roles. These data suggest that scavenger receptors function as co-receptors along with TLRs for HMGB1 in M1-type inflammatory macrophages.
Asunto(s)
Colitis/inmunología , Macrófagos/fisiología , Receptores Inmunológicos/metabolismo , Receptores Depuradores de Clase A/metabolismo , Sepsis/inmunología , Animales , Bovinos , Diferenciación Celular , Células Cultivadas , Colitis/inducido químicamente , Citocinas/metabolismo , Proteína HMGB1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores Inmunológicos/genética , Receptores Depuradores de Clase A/genética , Sepsis/inducido químicamente , Albúmina Sérica Bovina/administración & dosificación , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-ß and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.
Asunto(s)
Ácido Ascórbico/farmacología , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/prevención & control , Isoantígenos/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Animales , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/terapia , Humanos , Inmunoterapia Adoptiva , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología , Tretinoina/farmacologíaRESUMEN
Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.
Asunto(s)
Colitis/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Inmunoterapia Adoptiva/métodos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Ácido Ascórbico/administración & dosificación , Colitis/inducido químicamente , Secuencia Conservada , Islas de CpG/genética , Desmetilación , Dioxigenasas , Inducción Enzimática , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Humanos , Hipoxia , Ratones , Subgrupos de Linfocitos T/trasplante , Linfocitos T Reguladores/trasplanteRESUMEN
Regulatory T cells (Tregs) are an essential cell subset for the maintenance of immune homeostasis. Foxp3 (Forkhead box P3) is the Treg master gene which is essential for immune suppressing activity. In addition, Tregs are characterized by a distinct pattern of gene expression, including upregulation of immune-suppressive genes and silencing of inflammatory genes. The molecular mechanisms of Treg development and maintenance have been intensively investigated. Tregs are characterized by expression of the transcription factor Foxp3. Several intronic enhancers and a promoter at the Foxp3 gene locus were shown to play important roles in Treg differentiation. The enhancers have been designated as conserved non-coding sequences (CNSs) 0, 1, 2, and 3. We showed that the transcription factors Nr4a and Smad2/3 are essential for the development of thymic Tregs and induced Tregs, respectively. Recently, Treg-specific DNA demethylation has been shown to play an important role in Treg stability. DNA demethylation of CNS2 has been implicated in Treg stability, and recent reports have revealed that the ten-eleven translocation (Tet) family of demethylation factor plays an important role in CpG demethylation at CNS2. This article reviews the recent progress on the roles of transcription factors and epigenetic modifications in the differentiation, maintenance, and function of Tregs.
Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Activación de Linfocitos , Linfocitos T Reguladores/inmunología , Timo/fisiología , Animales , Metilación de ADN , Humanos , Tolerancia Inmunológica , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Proteína Smad2/genéticaRESUMEN
Psoriasis is considered a Th17-type autoimmune skin inflammatory disease; however, involvement of an autoantigen-specific TCR has not been established. In this study, we show that psoriasis-like skin inflammation can be induced by autoreactive Th17 cells. We previously developed the desmoglein 3-specific TCR-transgenic (Dsg3H1) mouse, in which CD4⺠T cells recognize physiological epidermal autoantigen. T cells from Dsg3H1 mice were polarized into Th17 cells in vitro and then adoptively transferred into Rag2â»/â» mice. Dsg3H1-Th17 cells induced severe psoriasis-like skin inflammation within 2 wk after transfer in the tissues in which desmoglein 3 is expressed. Such pathology was not observed when wild-type Th17 cells or Th1-skewed Dsg3H1 T cells were transferred, and it was strongly suppressed by anti-IL-12/23 and anti-IL-17 Abs. Although IFN-γâº/IL-17⺠T cells accumulated in the skin lesions of mice that received Dsg3H1-Th17 cells, IFN-γ-deficient Dsg3H1-Th17 cells were fully pathogenic. These results demonstrate that cutaneous psoriasis-like immunopathology can be developed by epidermis-specific recognition of Th17 cells, which is strictly dependent on IL-17 but not IFN-γ.
Asunto(s)
Autoantígenos/inmunología , Psoriasis/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Piel/inmunología , Células Th17/inmunología , Traslado Adoptivo , Animales , Separación Celular , Dermatitis/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We describe the cribrimorph cheilostome bryozoan Cribrilina mutabilis n. sp., which we detected as an epibiont on eelgrass (Zostera marina) at Akkeshi, Hokkaido, northern Japan. This species shows three distinct zooid types during summer: the R (rib), I (intermediate), and S (shield) types. Evidence indicates that zooids commit to development as a given type, rather than transform from one type to another with age. Differences in the frontal spinocyst among the types appear to be mediated by a simple developmental mechanism, acceleration or retardation in the production of lateral costal fusions as the costae elongate during ontogeny. Colonies of all three types were identical, or nearly so, in partial nucleotide sequences of the mitochondrial COI gene (555-631 bp), suggesting that they represent a single species. Zooid types varied temporally in overall frequency in the population: colonies contained nearly exclusively R-type zooids in mid-June; predominantly I-type, or both R- and I-type, zooids in mid-July; and I-type, S-type, or both I- and S-type zooids (interspersed or in discrete bands) in mid- to late August. Reproduction occurred throughout the season, but peaked in July, with only R- and I-type zooids reproducing. Reproductive zooids bear a vestigial compound (tripartite) ooecium and brood internally; S-type zooids, first appearing in August, were non-reproductive, which suggests that they may serve as an overwintering stage. As this species is easily accessible, common, and simple in form, it is potentially useful as a model system for studying polyphenism at multiple levels (zooid, colony, and population) in the context of life-history adaptations.
Asunto(s)
Briozoos/fisiología , Zosteraceae/fisiología , Animales , Briozoos/ultraestructura , Ecosistema , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Especies Introducidas , Japón , Estadios del Ciclo de Vida , Especificidad de la EspecieRESUMEN
Most viruses possess strategies to circumvent host immune responses. The measles virus (MV) nonstructural C protein suppresses the interferon response, thereby allowing efficient viral growth, but its detailed mechanism has been unknown. We identified Shc Src homology 2 domain-binding protein 1 (SHCBP1) as one of the host proteins interacting with the C protein. Knockdown of SHCBP1 using a short-hairpin RNA greatly reduced MV growth. SHCBP1 was found to be required for viral RNA synthesis in the minigenome assay and to bind to the MV phosphoprotein, a subunit of the viral RNA polymerase. A stretch of 12 amino acid residues in the C protein were sufficient for SHCBP1 binding, and the peptide containing these 12 residues could suppress MV RNA synthesis, like the full-length C protein. The central region of SHCBP1 was found to bind to the C protein, as well as the phosphoprotein, but the two viral proteins did not compete for SHCBP1 binding. Our results indicate that the C protein modulates MV RNA polymerase activity by binding to the host protein SHCBP1. SHCBP1 may be exploited as a target of antiviral compounds.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Virus del Sarampión/fisiología , Virus del Sarampión/patogenicidad , Proteínas Adaptadoras de la Señalización Shc/fisiología , Proteínas no Estructurales Virales/fisiología , Línea Celular , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus del Sarampión/genética , Modelos Biológicos , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas Adaptadoras de la Señalización Shc/antagonistas & inhibidores , Proteínas Adaptadoras de la Señalización Shc/genética , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genéticaRESUMEN
Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1ß) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1ß. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca(2+) level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca(2+) did not reduce virus-induced IL-1ß secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca(2+) flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.
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Calcio/metabolismo , Infecciones por Cardiovirus/metabolismo , Proteínas Portadoras/metabolismo , Virus de la Encefalomiocarditis/metabolismo , Inflamasomas/metabolismo , Proteínas Virales/metabolismo , Animales , Infecciones por Cardiovirus/genética , Proteínas Portadoras/genética , Virus de la Encefalomiocarditis/genética , Células HEK293 , Células HeLa , Humanos , Inflamasomas/genética , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Virales/genética , Virión/genética , Virión/metabolismoRESUMEN
T cell exhaustion impairs tumor immunity and contributes to resistance against immune checkpoint inhibitors. The nuclear receptor subfamily 4 group A (NR4a) family of nuclear receptors plays a crucial role in driving T cell exhaustion. In this study, we observe that NR4a1 and NR4a2 deficiency in CD8+ tumor-infiltrating lymphocytes (TILs) results in potent tumor eradication and exhibits not only reduced exhaustion characteristics but also an increase in the precursors/progenitors of exhausted T (Pre-Tex) cell fraction. Serial transfers of NR4a1-/-NR4a2-/-CD8+ TILs into tumor-bearing mice result in the expansion of TCF1+ (Tcf7+) stem-like Pre-Tex cells, whereas wild-type TILs are depleted upon secondary transfer. NR4a1/2-deficient CD8+ T cells express higher levels of stemness/memory-related genes and illustrate potent mitochondrial oxidative phosphorylation. Collectively, these findings suggest that inhibiting NR4a in tumors represents a potent immuno-oncotherapy strategy by increasing stem-like Pre-Tex cells and reducing exhaustion of CD8+ T cells.
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Linfocitos T CD8-positivos , Neoplasias , Animales , Ratones , Linfocitos Infiltrantes de Tumor , Neoplasias/genética , Microambiente TumoralRESUMEN
BACKGROUND: Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified. METHODS: In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice. RESULTS: Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors. CONCLUSIONS: NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.
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Inmunoterapia Adoptiva , Mitocondrias , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Mitocondrias/metabolismo , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Receptores de Hormona Tiroidea/metabolismo , Receptores de Hormona Tiroidea/genética , Neoplasias/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Proteínas de Unión al ADN , Receptores de EsteroidesRESUMEN
Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-ß for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-ß and CDK inhibitor, efficiently suppressed TGF-ß-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-ß inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-ß. These data suggest that CDK inhibitors modulate TGF-ß-signaling pathways, which restore TGF-ß-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance.