RESUMEN
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.
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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 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: Dialysis patients are susceptible to developing severe coronavirus disease 2019 (COVID-19) due to hypoimmunity. Antibody titers against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) after the primary vaccinations are lower in hemodialysis (HD) patients than in healthy individuals. This study aimed to evaluate the effect of a SARS-CoV-2 booster vaccination in HD and peritoneal dialysis (PD) patients based on antibody titers and cellular and humoral immunity. METHODS: Participants of the control, HD, and PD groups were recruited from 12 facilities. SARS-CoV-2 antigen-specific cytokine and IgG-antibody levels were measured. Regulatory T cells and memory B cells were counted using flow cytometry at 6 months after primary vaccination with BNT162b2 and 3 weeks after the booster vaccination in HD and PD patients and compared with those of a control group. RESULTS: Booster vaccination significantly enhanced the levels of antibodies, cytokines, and memory B cells in three groups. The HD group showed significantly higher levels of IgG-antibodies, IL-1ß, IL-2, IL-4, IL-17, and memory B cells than those in the control group at 3 weeks after the booster dose. The PD group tended to show similar trends to HD patients but had similar levels of IgG-antibodies, cytokines, and memory B cells to the control group. CONCLUSIONS: HD patients had significantly stronger cellular and humoral immune responses than the control 3 weeks after the booster dose. Our findings will help in developing better COVID-19 vaccination strategies for HD and PD patients.
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Anticuerpos Antivirales , Vacuna BNT162 , COVID-19 , Inmunidad Humoral , Inmunización Secundaria , Diálisis Renal , Humanos , Masculino , Femenino , COVID-19/inmunología , COVID-19/prevención & control , Persona de Mediana Edad , Anciano , Anticuerpos Antivirales/sangre , Vacuna BNT162/inmunología , Citocinas/sangre , SARS-CoV-2/inmunología , Vacunas contra la COVID-19/inmunología , Inmunidad Celular , Inmunoglobulina G/sangre , Japón , Células B de Memoria/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Diálisis Peritoneal , Pueblos del Este de AsiaRESUMEN
Interleukin (IL)-6 is abundantly expressed in the tumor microenvironment and is associated with poor patient outcomes. Here, we demonstrate that the deletion of the suppressor of cytokine signaling 3 (SOCS3) in T cells potentiates anti-tumor immune responses by conferring the anti-tumorigenic function of IL-6 in mouse and human models. In Socs3-deficient CD8+ T cells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumor effector function of T cells, while also modifying mitochondrial fitness to increase mitochondrial membrane potential and reactive oxygen species (ROS) levels and to promote metabolic glycolysis in the energy state. Furthermore, Socs3 deficiency reduces regulatory T cells and increases T helper 1 (Th1) cells. SOCS3 knockdown in human chimeric antigen receptor T (CAR-T) cells exhibits a strong anti-tumor response in humanized mice. Thus, genetic disruption of SOCS3 offers an avenue to improve the therapeutic efficacy of adoptive T cell therapy.
RESUMEN
Coronavirus disease 2019 (COVID-19) following primary immunization (breakthrough infection) has been reported in hemodialysis patients; however, their post-infection immune status remains unclear. We evaluated the humoral and cellular immunity of hemodialysis patients after breakthrough infection. Hemodialysis patients who had received primary immunization against COVID-19 at least six months prior to the study but developed mild/moderate COVID-19 before a booster dose (breakthrough infection group) and hemodialysis patients who were not infected with COVID-19 but received a booster dose (booster immunization group) were recruited. In both groups, SARS-CoV-2 antigen-specific cytokines and IgG levels were measured three weeks after infection or three weeks after receiving a booster dose. Memory T and B cells were also counted in the breakthrough infection group using flow cytometry three weeks after infection. Significantly higher SARS-CoV-2 antigen-specific IgG, IFN-γ, IL-5, TNF-α, and IL-6 levels occurred in the breakthrough infection group compared to the booster immunization group (p = 0.013, 0.039, 0.024, 0.017, and 0.039, respectively). The SARS-CoV-2 antigen-specific IgG and cytokine levels were not significantly different between the two groups. The breakthrough infection group had significantly higher percentages of central and effector memory T cells and regulatory T cells than the comparison group (p = 0.008, 0.031, and 0.026, respectively). Breakthrough infections may induce stronger cellular and humoral immune responses than booster immunizations in hemodialysis patients.
RESUMEN
Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.
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COVID-19 , Células B de Memoria , Humanos , SARS-CoV-2 , Células T de Memoria , Vacuna BNT162 , VacunaciónRESUMEN
Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.
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Proteínas de Unión al ADN/metabolismo , Desmogleína 3/metabolismo , Pénfigo/inmunología , Abatacept/farmacología , Traslado Adoptivo , Animales , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Desmogleína 3/genética , Antagonistas de Estrógenos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Masculino , Ratones , Ratones Noqueados , Linfocitos T Reguladores , Tamoxifeno/farmacologíaRESUMEN
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.
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Citocinas/inmunología , Tolerancia Inmunológica/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Animales , Humanos , Transducción de Señal/inmunologíaRESUMEN
T cells with a stem cell memory (TSCM) phenotype provide long-term and potent antitumor effects for T-cell transfer therapies. Although various methods for the induction of TSCM-like cells in vitro have been reported, few methods generate TSCM-like cells from effector/exhausted T cells. We have reported that coculture with the Notch ligand-expressing OP9 stromal cells induces TSCM-like (iTSCM) cells. Here, we established a feeder-free culture system to improve iTSCM cell generation from expanded chimeric antigen receptor (CAR)-expressing T cells; culturing CAR T cells in the presence of IL7, CXCL12, IGF-I, and the Notch ligand, hDLL1. Feeder-free CAR-iTSCM cells showed the expression of cell surface markers and genes similar to that of OP9-hDLL1 feeder cell-induced CAR-iTSCM cells, including the elevated expression of SCM-associated genes, TCF7, LEF1, and BCL6, and reduced expression of exhaustion-associated genes like LAG3, TOX, and NR4A1. Feeder-free CAR-iTSCM cells showed higher proliferative capacity depending on oxidative phosphorylation and exhibited higher IL2 production and stronger antitumor activity in vivo than feeder cell-induced CAR-iTSCM cells. Our feeder-free culture system represents a way to rejuvenate effector/exhausted CAR T cells to SCM-like CAR T cells. Significance: Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.
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Células Madre , Linfocitos T , Ligandos , Inmunoterapia Adoptiva/métodos , Técnicas de CocultivoRESUMEN
Recent studies have shown that stem cell memory T (TSCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, "CAR-iTSCM" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM formation is an effective strategy for improving cancer immunotherapy. SIGNIFICANCE: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.
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Proteína Forkhead Box M1/metabolismo , Memoria Inmunológica/inmunología , Leucemia/inmunología , Biogénesis de Organelos , Receptores Quiméricos de Antígenos/inmunología , Receptores Notch/metabolismo , Linfocitos T/inmunología , Animales , Diferenciación Celular , Técnicas de Cocultivo , Humanos , Inmunoterapia Adoptiva , Leucemia/metabolismo , Leucemia/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción de Señal , Células Madre/inmunología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
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.
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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
Inhibition of immune checkpoint molecules, PD-1 and CTLA4, has been shown to be a promising cancer treatment. PD-1 and CTLA4 inhibit TCR and co-stimulatory signals. The third T cell activation signal represents the signals from the cytokine receptors. The cytokine interferon-γ (IFNγ) plays an important role in anti-tumor immunity by activating cytotoxic T cells (CTLs). Most cytokines use the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and the suppressors of cytokine signaling (SOCS) family of proteins are major negative regulators of the JAK/STAT pathway. Among SOCS proteins, CIS, SOCS1, and SOCS3 proteins can be considered the third immunocheckpoint molecules since they regulate cytokine signals that control the polarization of CD4+ T cells and the maturation of CD8+ T cells. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in terms of their anti-tumor immunity and potential applications.
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Inmunoterapia/métodos , Neoplasias/terapia , Proteína 1 Supresora de la Señalización de Citocinas/inmunología , Proteína 3 Supresora de la Señalización de Citocinas/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Animales , Humanos , Modelos Inmunológicos , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides.
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Proteína Morfogenética Ósea 2 , Regeneración Ósea/efectos de los fármacos , Diferenciación Celular , Oligopéptidos/farmacología , Osteoblastos/efectos de los fármacos , Animales , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Oligopéptidos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/fisiología , Unión Proteica , Ligando RANK/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Tooth extraction causes bone resorption of the alveolar bone volume. Although recombinant human bone morphogenetic protein 2 (rhBMP-2) markedly promotes de novo bone formation after tooth extraction, the application of high-dose rhBMP-2 may induce side effects, such as swelling, seroma, and an increased cancer risk. Therefore, reduction of the necessary dose of rhBMP-2 which can still obtain sufficient bone mass is necessary by developing a new osteogenic reagent. Recently, we showed that the systemic administration of OP3-4 peptide, which was originally designed as a bone resorption inhibitor, had osteogenic ability both in vitro and in vivo. This study evaluated the ability of the local application of OP3-4 peptide to promote bone formation in a murine tooth extraction model with a very low-dose of BMP. The mandibular incisor was extracted from 10-week-old C57BL6/J male mice and a gelatin hydrogel containing rhBMP-2 with or without OP3-4 peptide (BMP/OP3-4) was applied to the socket of the incisor. Bone formation inside the socket was examined radiologically and histologically at 21 days after the extraction. The BMP/OP3-4-group showed significant bone formation inside the mandibular extraction socket compared to the gelatin-hydrogel-carrier-control group or rhBMP-2-applied group. The BMP/OP3-4-applied mice showed a lower reduction of alveolar bone and fewer osteoclast numbers, suggesting that the newly formed bone inside the socket may prevent resorption of the cortical bone around the extraction socket. Our data revealed that OP3-4 peptide promotes BMP-mediated bone formation inside the extraction socket of mandibular bone, resulting in preservation from the loss of alveolar bone.
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Pérdida de Hueso Alveolar/etiología , Pérdida de Hueso Alveolar/prevención & control , Oligopéptidos/farmacología , Osteogénesis/efectos de los fármacos , Extracción Dental/efectos adversos , Pérdida de Hueso Alveolar/fisiopatología , Animales , Proteína Morfogenética Ósea 2/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Incisivo/cirugía , Masculino , Ratones , Alveolo Dental/efectos de los fármacos , Alveolo Dental/fisiologíaRESUMEN
INTRODUCTION: We designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis. METHODS: Twenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed. RESULTS: The OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites. CONCLUSION: The peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.
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Artritis Experimental/tratamiento farmacológico , Resorción Ósea/prevención & control , Cartílago Articular/efectos de los fármacos , Oligopéptidos/farmacología , Ligando RANK/metabolismo , Secuencia de Aminoácidos , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Densidad Ósea/efectos de los fármacos , Resorción Ósea/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/sangre , Ensayo de Inmunoadsorción Enzimática , Infusiones Subcutáneas , Masculino , Ratones Endogámicos DBA , Oligopéptidos/administración & dosificación , Oligopéptidos/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoprotegerina/administración & dosificación , Osteoprotegerina/metabolismo , Osteoprotegerina/farmacología , Péptidos/sangre , Unión Proteica , Tibia/efectos de los fármacos , Tibia/metabolismo , Tibia/patología , Microtomografía por Rayos XRESUMEN
The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. In this study, we examined the fate of thymic Tregs in TNF-α/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1-null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1, which is reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remained functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7Rα, which is a possible marker of late-stage mature Tregs. Finally, TA-KO fetal liver chimeric mice developed a neuropilin-1(+) splenic Treg population from TA-KO cells, suggesting that Treg arrest was caused by a lack of RelA in the thymic environment. Taken together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment.
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Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timo/inmunología , Timo/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Biomarcadores , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Islas de CpG , Metilación de ADN , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Sitios Genéticos , Masculino , Ratones , Ratones Noqueados , Fenotipo , Receptores de Interleucina-7/metabolismo , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Factor de Transcripción ReIA/genéticaRESUMEN
Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100.
Asunto(s)
Células Dendríticas/metabolismo , Interleucina-23/metabolismo , Subunidad p52 de NF-kappa B/fisiología , Proteínas Proto-Oncogénicas c-rel/antagonistas & inhibidores , Animales , Secuencia de Bases , Cartilla de ADN , Células HEK293 , Humanos , Ratones , Subunidad p52 de NF-kappa B/genética , Reacción en Cadena de la PolimerasaRESUMEN
Bone remodeling and hematopoiesis are interrelated and bone marrow (BM) macrophages are considered to be important for both bone remodeling and maintenance of the hematopoietic niche. We found that NF-κB Rela-deficient chimeric mice, generated by transplanting Rela (-/-) fetal liver cells into lethally irradiated hosts, developed severe osteopenia, reduced lymphopoiesis and enhanced mobilization of hematopoietic stem and progenitor cells when BM cells were completely substituted by Rela-deficient cells. Rela (-/-) hematopoietic stem cells from fetal liver had normal hematopoietic ability, but those harvested from the BM of osteopenic Rela (-/-) chimeric mice had reduced repopulation ability, indicating impairment of the microenvironment for the hematopoietic niche. Osteopenia in Rela (-/-) chimeric mice was due to reduced bone formation, even though osteoblasts differentiated from host cells. This finding indicates impaired functional coupling between osteoblasts and hematopoietic stem cell-derived cells. Rela-deficient BM macrophages exhibited an aberrant inflammatory phenotype, and transplantation with wild-type F4/80(+) BM macrophages recovered bone formation and ameliorated lymphopoiesis in Rela (-/-) chimeric mice. Therefore, RELA in F4/80(+) macrophages is important both for bone homeostasis and for maintaining the hematopoietic niche after lethal irradiation and hematopoietic stem cell transplantation.
Asunto(s)
Hematopoyesis/genética , Macrófagos/metabolismo , Osteogénesis/genética , Nicho de Células Madre/genética , Factor de Transcripción ReIA/deficiencia , Animales , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Médula Ósea/metabolismo , Médula Ósea/patología , Trasplante de Células Madre Hematopoyéticas , Linfopoyesis/genética , Masculino , Ratones , Ratones Noqueados , Osteoclastos/metabolismo , Factor de Transcripción ReIA/genética , Quimera por Trasplante , Irradiación Corporal TotalRESUMEN
Although the NF-kappaB transcription factors participate in both innate and adaptive immune responses, little is known about the role of the RelA subunit because mice lacking the rela gene die at embryonic day 14. To elucidate the role of RelA in Leishmania major infection, we prepared fetal liver chimeric mice by adoptively transferring embryonic day 13.5 rela(-/-) or rela(+/+) fetal liver into lethally irradiated host mice. About 90% of the peripheral lymphocytes of the chimeric mice had differentiated from rela fetal liver cells. The rela(-/-) fetal liver chimeric mice were highly sensitive to infection with L. major and died within 11 wk after infection. Despite the severity of the disease, parasite Ag-reactive Th1 cells developed normally. The rela(-/-) macrophages were less able to control intracellular parasite replication than rela(+/+) macrophages, despite showing equally efficient phagocytosis. Both in vitro NO production of macrophages and in vivo expression of NO synthase 2 in the lesions and draining lymph nodes was reduced in rela(-/-) fetal liver chimeric mice. Moreover, up-regulation of Fas in rela(-/-) macrophages was impaired both after in vitro stimulation with LPS and after in vivo infection with L. major, implying a defect in their ability to eliminate infected cells. Thus, RelA is necessary for macrophages to be resistant to intracellular parasite infection.
Asunto(s)
Leishmania major/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Transcripción ReIA/metabolismo , Regulación hacia Arriba , Receptor fas/metabolismo , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Inducción Enzimática , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Células TH1/citología , Células TH1/inmunología , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/genética , Receptor fas/inmunologíaRESUMEN
IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a common p40 subunit is shared with IL-12. IL-23 promotes the inflammatory response by inducing the expansion of CD4(+) cells producing IL-17. The regulation of p19 gene expression has been less studied than that of p40 subunit expression, which in macrophages is well known to be dependent on NF-kappaB. To clarify the role of NF-kappaB in expression of the p19 gene, we analyzed mRNA levels in NF-kappaB-deficient macrophages. As reported to occur in dendritic cells, p19 expression was dramatically reduced in c-rel-deficient macrophages. Moreover, we found that p19 expression was halved in rela-deficient macrophages, but it was enhanced in p52-deficient macrophages. The p19 promoter contains three putative kappaB sites, located at nt -642 to -632 (kappaB-642), nt -513 to -503 (kappaB-513), and nt -105 to -96 (kappaB-105), between the transcription start site and -937 bp upstream in the p19 promoter region. Although EMSA analysis indicated that both kappaB-105 and kappaB-642, but not kappaB-513, bound to NF-kappaB in vitro, luciferase-based reporter assays showed that the most proximal kappaB site, kappaB-105, was uniquely indispensable to the induction of p19 transcription. Chromatin immunoprecipitation demonstrated in vivo association of RelA, c-Rel, and p50 with kappaB-105 of the p19 promoter. These results provide the evidence that the association of RelA and c-Rel with the proximal kappaB site in the p19 promoter is required to induce of p19 expression.