RESUMEN
Microglial cell activation is the earliest biomarker of the inflammatory processes that cause central nervous system (CNS) lesions in multiple sclerosis. We hypothesized that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by activated microglia and macrophages in the CNS inhibits these inflammatory processes. To test this hypothesis, we targeted the Cyp27b1 gene specifically in myeloid cells, then analyzed the influence of disrupted myeloid cell 1,25-(OH)2D3 synthesis on vitamin D3-mediated resistance to experimental autoimmune encephalomyelitis (EAE). Myeloid cell 1,25-(OH)2D3 synthesis was essential for vitamin D3-mediated EAE resistance. Increased CTLA-4 expression in the CNS-infiltrating CD4+ Tconv and Treg cells and decreased splenic B cell CD86 expression correlated with resistance. These new data provide solid support for the view that vitamin D3 reduces MS risk in part through a mechanism involving myeloid cell 1,25-(OH)2D3 production and CTLA-4 upregulation in CNS-infiltrating CD4+ T cells. We suggest that CTLA-4 serves as a vitamin D3-regulated immunological checkpoint in multiple sclerosis prevention.
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Linfocitos T CD4-Positivos/inmunología , Antígeno CTLA-4/análisis , Calcitriol/biosíntesis , Colecalciferol/farmacología , Encefalomielitis Autoinmune Experimental/prevención & control , Macrófagos/metabolismo , Microglía/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Animales , Antígeno B7-2/análisis , Antígeno CTLA-4/fisiología , Modelos Animales de Enfermedad , Femenino , Ratones , Esclerosis Múltiple/prevención & control , Elemento de Respuesta a la Vitamina D/fisiologíaRESUMEN
Metabolically healthy obesity (MHO) accounts for roughly 35% of all obese patients. There is no clear consensus that has been reached on whether MHO is a stable condition or merely a transitory period between metabolically healthy lean and metabolically unhealthy obesity (MUO). Additionally, the mechanisms underlying MHO and any transition to MUO are not clear. Macrophages are the most common immune cells in adipose tissues and have a significant presence in atherosclerosis. Fas (or CD95), which is highly expressed on macrophages, is classically recognized as a pro-apoptotic cell surface receptor. However, Fas also plays a significant role as a pro-inflammatory molecule. Previously, we established a mouse model (ApoE-/-/miR155-/-; DKO mouse) of MHO, based on the criteria of not having metabolic syndrome (MetS) and insulin resistance (IR). In our current study, we hypothesized that MHO is a transition phase toward MUO, and that inflammation driven by our newly classified CD95+CD86- macrophages is a novel mechanism for this transition. We found that, with extended (24 weeks) high-fat diet feeding (HFD), MHO mice became MUO, shown by increased atherosclerosis. Mechanistically, we found the following: 1) at the MHO stage, DKO mice exhibited increased pro-inflammatory markers in adipose tissue, including CD95, and serum; 2) total adipose tissue macrophages (ATMs) increased; 3) CD95+CD86- subset of ATMs also increased; and 4) human aortic endothelial cells (HAECs) were activated (as determined by upregulated ICAM1 expression) when incubated with conditioned media from CD95+-containing DKO ATMs and human peripheral blood mononuclear cells-derived macrophages in comparison to respective controls. These results suggest that extended HFD in MHO mice promotes vascular inflammation and atherosclerosis via increasing CD95+ pro-inflammatory ATMs. In conclusion, we have identified a novel molecular mechanism underlying MHO transition to MUO with HFD. We have also found a previously unappreciated role of CD95+ macrophages as a potentially novel subset that may be utilized to assess pro-inflammatory characteristics of macrophages, specifically in adipose tissue in the absence of pro-inflammatory miR-155. These findings have provided novel insights on MHO transition to MUO and new therapeutic targets for the future treatment of MUO, MetS, other obese diseases, and type II diabetes.
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Inflamación/inmunología , Macrófagos/fisiología , MicroARNs/fisiología , Obesidad Metabólica Benigna/inmunología , Receptor fas/análisis , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Aorta , Enfermedades de la Aorta/etiología , Aterosclerosis/etiología , Antígeno B7-2/análisis , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Dieta Alta en Grasa/efectos adversos , Progresión de la Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Inflamación/complicaciones , Molécula 1 de Adhesión Intercelular/biosíntesis , Macrófagos/química , Macrófagos/clasificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Obesidad Metabólica Benigna/metabolismo , Obesidad Metabólica Benigna/patología , Vasculitis/etiologíaRESUMEN
Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.
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Antígeno B7-2/análisis , Rastreo Celular/métodos , Proteínas Luminiscentes/análisis , Microscopía Fluorescente/métodos , Animales , Antígeno B7-2/genética , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Células HeLa , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Mutación , Procesos Fotoquímicos , Conformación ProteicaRESUMEN
Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.
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Presentación de Antígeno/inmunología , Eritroblastos/inmunología , Eritroblastos/virología , Septicemia Hemorrágica Viral/inmunología , Septicemia Hemorrágica Viral/virología , Novirhabdovirus/inmunología , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/virología , Animales , Presentación de Antígeno/genética , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Antígenos CD/análisis , Antígenos CD/inmunología , Autofagosomas/efectos de los fármacos , Autofagosomas/inmunología , Autofagosomas/virología , Autofagia/efectos de los fármacos , Autofagia/inmunología , Antígeno B7-2/análisis , Antígeno B7-2/inmunología , Biomarcadores/análisis , Septicemia Hemorrágica Viral/genética , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunoglobulinas/análisis , Inmunoglobulinas/inmunología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/inmunología , Niclosamida/farmacología , Proteínas de la Nucleocápside , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteómica , Proteína Sequestosoma-1/metabolismo , Antígeno CD83RESUMEN
Friend virus (FV) is a naturally occurring mouse retrovirus that infects dividing cells of the hematopoietic lineage, including antigen-presenting cells (APCs). The infection of APCs by viruses often induces their dysfunction, and it has been shown that FV infection reduces the ability of dendritic cells (DCs) to prime critical CD8+ T cell responses. Nonetheless, mice mount vigorous CD8+ T cell responses, so we investigated whether B cells might serve as alternative APCs during FV infection. Direct ex vivo analysis of B cells from FV-infected mice revealed that infected but not uninfected B cells upregulated expression of the costimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC-II) molecules. Furthermore, in vitro studies showed that, compared to uninfected B cells from the same mice, the FV-infected B cells had significantly enhanced APC function, as measured by their capacity to prime CD8+ T cell activation and proliferation. Thus, in contrast to DCs, infection of B cells with FV enhanced their APC capacity and ability to stimulate the CD8+ T cell responses essential for virus control. FV infections also induce the activation and expansion of regulatory T cells (Tregs), so it was of interest to determine the impact of Tregs on B cell activation. The upregulation of costimulatory molecule expression and APC function of B cells was even more strongly enhanced by in vivo depletion of regulatory T cells than infection. Thus, Tregs exert potent homeostatic suppression of B cell activation that is partially overcome by FV infection.IMPORTANCE The primary role of B cells in immunity is considered the production of pathogen-specific antibodies, but another, less-well-studied, function of B cells is to present foreign antigens to T cells to stimulate their activation and proliferation. Dendritic cells (DCs) are considered the most important antigen-presenting cells (APCs) for CD8+ T cells, but DCs lose APC function when infected with Friend virus (FV), a model retrovirus of mice. Interestingly, B cells were better able to stimulate CD8+ T cell responses when they were infected with FV. We also found that the activation status of B cells under homeostatic conditions was potently modulated by regulatory T cells. This study illustrates an important link between B cell and T cell responses and illustrates an additional mechanism by which regulatory T cells suppress critical T cell responses during viral infections.
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Presentación de Antígeno , Linfocitos B/inmunología , Virus de la Leucemia Murina de Friend/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos B/química , Antígeno B7-1/análisis , Antígeno B7-2/análisis , Antígenos CD40/análisis , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Antígenos de Histocompatibilidad Clase II/análisis , Leucemia Experimental/inmunología , Leucemia Experimental/virología , Activación de Linfocitos , Ratones , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virologíaRESUMEN
For the treatment of metastatic renal cell cancer several strategies are used among which the mTOR inhibitor everolimus. As mTOR plays an important role in the immune system, e.g., by controlling the expression of the transcription factor FoxP3 thereby regulating regulatory T cells (Tregs), it plays a key role in the balance between tolerance and inflammation. Previous reports showed stimulatory effects of mTOR inhibition on the expansion of Tregs, an effect that can be considered detrimental in terms of cancer control. Since metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs, a phase 1 clinical trial was conducted to comprehensively investigate the immune-modulating effects of several dosages and schedules of CTX in combination with the standard dose of everolimus, with the explicit aim to achieve selective Treg depletion. Our data show that 50 mg of CTX once daily and continuously administered, in combination with the standard dose of 10 mg everolimus once daily, not only results in depletion of Tregs, but also leads to a reduction in MDSC, a sustained level of the CD8+ T-cell population accompanied by an increased effector to suppressor ratio, and reversal of negative effects on three peripheral blood DC subsets. These positive effects on the immune response may contribute to improved survival, and therefore this combination therapy is further evaluated in a phase II clinical trial.
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Linfocitos T CD8-positivos/efectos de los fármacos , Carcinoma de Células Renales/inmunología , Ciclofosfamida/farmacología , Everolimus/farmacología , Neoplasias Renales/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Antígeno B7-2/análisis , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Metástasis de la Neoplasia , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (pâ¯=â¯0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (pâ¯=â¯0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.
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5'-Nucleotidasa/biosíntesis , Antígeno B7-2/biosíntesis , Biomarcadores de Tumor/análisis , Neuropilina-1/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , 5'-Nucleotidasa/análisis , Antígeno B7-2/análisis , Niño , Preescolar , Femenino , Proteínas Ligadas a GPI/análisis , Proteínas Ligadas a GPI/biosíntesis , Humanos , Masculino , Neoplasia Residual , Neuropilina-1/análisis , Células Precursoras de Linfocitos B/patologíaRESUMEN
Understanding host immune pathways associated with tissue damage during reactions are of upmost importance to the development of immune intervention strategies. The participation of monocytes in leprosy reactions was evaluated by determining the frequency of monocyte subsets and the degree of cellular activation through the expression of MHCII and the co-stimulatory molecules CD40, CD80, CD86. Leprosy subjects with or without reactions were included in this cross-sectional study. Peripheral blood mononuclear cell were isolated and stained ex vivo to determine monocyte subsets and the degree of cellular activation by flow cytometry. Intermediate monocytes were increased in leprosy patients with reactions when compared to patients without reactions. Although no difference was detected in the frequency of monocyte subsets between type 1 and 2 reactions, the expression of CD80 was increased in monocytes from patients with type 1 reactions and CD40 was higher in paucibacillary subjects presenting type 1 reactions. Moreover, CD86 and MHC II expression were higher in intermediate monocytes when compared to the other subsets in leprosy reaction types 1 and 2. Intermediate monocyte activation with CD86 and MHCII expression is involved with both type 1 and 2 reactions, whereas CD80 and CD40 expression is related to type 1 reactions.
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Antígeno B7-1/análisis , Antígeno B7-2/análisis , Antígenos CD40/análisis , Lepra/inmunología , Adolescente , Adulto , Anciano , Presentación de Antígeno , Biomarcadores/análisis , Estudios Transversales , Femenino , Citometría de Flujo , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Adulto JovenRESUMEN
Dendritic cells (DCs) are critical initiators of innate immunity in the kidney and orchestrate inflammation following ischemia-reperfusion injury. The role of the mammalian/mechanistic target of rapamycin (mTOR) in the pathophysiology of renal ischemia-reperfusion injury has been characterized. However, the influence of DC-based alterations in mTOR signaling is unknown. To address this, bone marrow-derived mTORC2-deficient (Rictor-/-) DCs underwent hypoxia-reoxygenation and then analysis by flow cytometry. Adoptive transfer of wild-type or Rictor-/- DC to C57BL/6 mice followed by unilateral or bilateral renal ischemia-reperfusion injury (20 min ischemia) was used to assess their in vivo migratory capacity and influence on tissue injury. Age-matched male DC-specific Rictor-/- mice or littermate controls underwent bilateral renal ischemia-reperfusion, followed by assessment of renal function, histopathology, and biomolecular and cell infiltration analysis. Rictor-/- DCs expressed more costimulatory CD80/CD86 but less coinhibitory programmed death ligand 1 (PDL1), a pattern that was enhanced by hypoxia-reoxygenation. They also demonstrated enhanced migration to the injured kidney and induced greater tissue damage. Following ischemia-reperfusion, Rictor-/- DC mice developed higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production compared to littermate controls. Additionally, a greater influx of both neutrophils and T cells was seen in Rictor-/- DC mice, along with CD11c+MHCII+CD11bhiF4/80+ renal DC, that expressed more CD86 but less PDL1. Thus, DC-targeted elimination of Rictor enhances inflammation and migratory responses to the injured kidney, highlighting the regulatory roles of both DCs and Rictor in the pathophysiology of acute kidney injury.
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Lesión Renal Aguda/etiología , Células Dendríticas/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina/fisiología , Animales , Antígeno B7-2/análisis , Citocinas/genética , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/deficiencia , Ratones Endogámicos C57BL , Infiltración Neutrófila , Transducción de Señal/fisiologíaRESUMEN
Delayed-type hypersensitivity (DTH) reactions are among the common reasons for drug withdrawal from clinical use during the post-marketing stage. Several in vivo methods have been developed to test DTH responses in animal models. They include the local lymph node assay (LLNA) and local lymph node proliferation assay (LLNP). While LLNA is instrumental in testing topically administered formulations (e.g., creams), the LLNP was proven to be predictive of drug-mediated DTH in response to small molecule pharmaceuticals. Global efforts in reducing the use of research animals lead to the development of in vitro models to predict test-material-mediated DTH. Two such models include analysis of surface marker expression in human cell lines THP-1 and U-937. These tests are known as the human cell line activation test (hCLAT) and myeloid U937 skin sensitization test (MUSST or U-SENS), respectively. Here we describe experimental procedures for all these methods, discuss their in vitro-in vivo correlation, and suggest a strategy for applying these tests to analyze engineered nanomaterials and nanotechnology-formulated drug products.
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Alérgenos/efectos adversos , Citometría de Flujo/métodos , Hipersensibilidad Tardía/etiología , Nanopartículas/efectos adversos , Pruebas Cutáneas/métodos , Animales , Antígeno B7-2/análisis , Línea Celular , Proliferación Celular , Femenino , Humanos , Antígenos Comunes de Leucocito/análisis , Leucocitos/citología , Ensayo del Nódulo Linfático Local , Masculino , Ratones Endogámicos BALB C , Células THP-1RESUMEN
Microglia activation and neuroinflammation are common features of neurodegenerative conditions, including alcohol use disorders (AUDs). When activated, microglia span a continuum of diverse phenotypes ranging from classically activated, pro-inflammatory (M1) microglia/macrophages to alternatively activated, growth-promoting (M2) microglia/macrophages. Identifying microglia phenotypes is critical for understanding the role of microglia in the pathogenesis of AUDs. Therefore, male rats were gavaged with 25% (w/v) ethanol or isocaloric control diet every 8 h for 4 days and sacrificed at 0, 2, 4, and 7 days after alcohol exposure (e.g., T0, T2, etc.). Microglia were isolated from hippocampus and entorhinal cortices by Percoll density gradient centrifugation. Cells were labeled with microglia surface antigens and analyzed by flow cytometry. Consistent with prior studies, isolated cells yielded a highly enriched population of brain macrophages/microglia (>95% pure), evidenced by staining for the macrophage/microglia antigen CD11b. Polarization states of CD11b+CD45low microglia were evaluated by expression of M1 surface markers, major histocompatibility complex (MHC) II, CD32, CD86, and M2 surface marker, CD206 (mannose receptor). Ethanol-treated animals begin to show increased expression of M1 and M2 markers at T0 (p = n.s.), with significant changes at the T2 time point. At T2, expression of M1 markers, MHC-II, CD86, and CD32 were increased (p < 0.05) in hippocampus and entorhinal cortices, while M2 marker, CD206, was increased significantly only in entorhinal cortices (p < 0.05). All effects resolved to control levels by T4. In summary, four-day binge alcohol exposure produces a transient increase in both M1 (MHC-II, CD32, and CD86) and M2 (CD206) populations of microglia isolated from the entorhinal cortex and hippocampus. Thus, these findings that both pro-inflammatory and potentially beneficial, recovery-promoting microglia phenotypes can be observed after a damaging exposure of alcohol are critically important to our understanding of the role of microglia in the pathogenesis of AUDs.
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Etanol/administración & dosificación , Microglía/efectos de los fármacos , Fenotipo , Alcoholismo/fisiopatología , Animales , Antígeno B7-2/análisis , Biomarcadores/análisis , Corteza Entorrinal/citología , Hipocampo/citología , Antígenos de Histocompatibilidad Clase II/análisis , Lectinas Tipo C/análisis , Activación de Macrófagos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/análisis , Microglía/clasificación , Microglía/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/análisis , Receptores de IgG/análisisRESUMEN
Objective To promote the induction and separation efficiency of bone marrow-derived dendritic cells (BMDCs) in vitro through optimizing the inducing and isolating process by multiple cytokines. Methods The factors to be optimized in single factor tests included recombinant mouse granulocyte macrophage-colony stimulating factor (rmGM-CSF), recombinant mouse interleukine 4 (rmIL-4), lipopolysaccharide (LPS), recombinant mouse tumor necrosis factor α (rmTNF-α) and inducing time. The numbers of immature dendritic cells (imDCs) and mature dendritic cells (mDCs) were investigated as the indicators. Box-Behnken experimental design-response surface methodology was used to analyze and verify the data. Morphological changes were observed using the inverted microscopy and the transmission electron microscopy. Surface molecules including CD11c and CD86 were detected using the flow cytometry. Results The optimum inducing conditions for imDCs were obtained as follows: rmGM-CSF was 46 ng/mL, rmIL-4 was 24 ng/mL, inducing time was 6 days, and the number of imDCs was (4.58±0.28)×106 cells, and the relative deviation was 4.00%. The optimum inducing conditions for mDCs were as follows: LPS was 1.4 µg/mL, rmTNF-α was 30 ng/mL, inducing time was 1 day, and the number of mDCs was (4.21±0.15)×106 cells, and the relative deviation was 3.80%. Sufficient typical imDCs and mDCs were obtained within 5-7 days of induction in vitro. Also, flow cytometry showed that the amplified imDCs had a high expression of CD11c (68.62%±2.3%) and a low expression of CD86 (37.95%±1.8%), and the mDCs had high expressions of both CD86 (90.34%±1.4%) and CD11c (82.05%±1.6%). Conclusion The combination of single factor tests and Box-Behnken design -response surface methodology could optimize the inducing and isolating method for DCs in vitro by multiple cytokines rapidly and efficiently, which provided basic experiment materials for further studies.
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Separación Celular/métodos , Citocinas/farmacología , Células Dendríticas/fisiología , Animales , Antígeno B7-2/análisis , Antígeno CD11c/análisis , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Cytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection. METHODS: Mouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells. RESULTS: This assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection. CONCLUSIONS: The findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV.
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Antígeno CD11c/análisis , Células Dendríticas/química , Infecciones por Herpesviridae/veterinaria , Células Asesinas Naturales/química , Pulmón/inmunología , Muromegalovirus/inmunología , Linfocitos T/química , Animales , Antígeno B7-2/análisis , Sangre/inmunología , Femenino , Citometría de Flujo , Infecciones por Herpesviridae/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Inmunofenotipificación , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/inmunologíaAsunto(s)
Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Ginsenósidos/farmacología , Hematopoyesis/efectos de los fármacos , Anemia Aplásica/tratamiento farmacológico , Animales , Antígeno B7-2/análisis , Células Dendríticas/citología , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBARESUMEN
Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D-related (HLA-DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA-DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2 weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over-represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.
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Enfermedades Autoinmunes/inmunología , Macrófagos/inmunología , Receptores de IgG/análisis , Receptores de IgG/genética , Bazo/inmunología , Trombocitopenia/inmunología , Adulto , Anciano , Enfermedades Autoinmunes/cirugía , Enfermedades Autoinmunes/terapia , Antígeno B7-2/análisis , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina G/sangre , Inmunoglobulinas Intravenosas/uso terapéutico , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Fagocitosis , Fenotipo , Polimorfismo Genético , Receptores de IgG/inmunología , Bazo/citología , Esplenectomía , Trombocitopenia/cirugía , Trombocitopenia/terapiaRESUMEN
The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Owing to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole-lung bronchoalveolar lavage. Six subsets of phagocytic APC (HLA-DR+) were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1-CD14+, BDCA1+CD14+, BDCA1+CD14-, and BDCA1-CD14- cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared with E. coli Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared with the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole-genome transcriptional profiling revealed a clade of "true dendritic cells" consisting of Langerin+, BDCA1+CD14+, and BDCA1+CD14- cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6 Each clade, and each member of both clades, was discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.
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Células Dendríticas/fisiología , Pulmón/inmunología , Macrófagos Alveolares/fisiología , Macrófagos/fisiología , Adulto , Anciano , Antígenos CD/análisis , Antígenos CD1/análisis , Antígeno B7-2/análisis , Células Dendríticas/clasificación , Perfilación de la Expresión Génica , Glicoproteínas/análisis , Humanos , Inmunoglobulinas/análisis , Receptores de Lipopolisacáridos/análisis , Pulmón/microbiología , Macrófagos/clasificación , Glicoproteínas de Membrana/análisis , Persona de Mediana Edad , Antígeno CD83RESUMEN
Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers. We found that B cells expressing the activation markers CD86, CD80, PD1, and CD40L, as well as CD19+CD27-IgD- double-negative B cells, were enriched in African American patients vs. patients of European ancestry. In addition to increased expression of CD40L, surface levels of CD40 on B cells were lower, suggesting the engagement of the CD40 pathway. In vitro experiments confirmed that CD40L expressed by B cells could lead to CD40 activation and internalization on adjacent B cells. To conclude, these results indicate that, compared with European American patients, African American SLE patients present with a particularly active B cell component, possibly via the activation of the CD40/CD40L pathway. These data may help guide the development of novel therapies.
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Linfocitos B/citología , Lupus Eritematoso Sistémico/etnología , Negro o Afroamericano , Antígenos de Superficie/análisis , Antígeno B7-2/análisis , Antígenos CD40/análisis , Ligando de CD40/análisis , Humanos , FenotipoRESUMEN
CD4(+)Foxp3(+) regulatory T cells (Tregs) are key immune suppressors that regulate immunity in diverse tissues. The tissue and/or inflammatory signals that influence the magnitude of the Treg response remain unclear. To define signals that promote Treg accumulation, we developed a simple system of skin inflammation using defined Ags and adjuvants that induce distinct cytokine milieus: OVA protein in CFA, aluminum salts (Alum), and Schistosoma mansoni eggs (Sm Egg). Polyclonal and Ag-specific Treg accumulation in the skin differed significantly between adjuvants. CFA and Alum led to robust Treg accumulation, with >50% of all skin CD4(+) T cells being Foxp3(+) In contrast, Tregs accumulated poorly in the Sm Egg-inflamed skin. Surprisingly, we found no evidence of inflammation-specific changes to the Treg gene program between adjuvant-inflamed skin types, suggesting a lack of selective recruitment or adaptation to the inflammatory milieu. Instead, Treg accumulation patterns were linked to differences in CD80/CD86 expression by APC and the regulation of CD25 expression, specifically in the inflamed skin. Inflammatory cues alone, without cognate Ag, differentially supported CD25 upregulation (CFA and Alum > Sm Egg). Only in inflammatory milieus that upregulated CD25 did the provision of Ag enhance local Treg proliferation. Reduced IL-33 in the Sm Egg-inflamed environment was shown to contribute to the failure to upregulate CD25. Thus, the magnitude of the Treg response in inflamed tissues is controlled at two interdependent levels: inflammatory signals that support the upregulation of the important Treg survival factor CD25 and Ag signals that drive local expansion.
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Dermatitis/inmunología , Subunidad alfa del Receptor de Interleucina-2/fisiología , Activación de Linfocitos , Piel/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos/inmunología , Antígeno B7-1/análisis , Antígeno B7-2/análisis , Femenino , Inflamación/inmunología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Mechanisms by which spontaneous clearance of acute hepatitis C occurs are unclear. A critical role for the innate immune system and IFNL4 polymorphisms has been proposed. This study investigates whether Toll-like receptor (TLR) expression and signaling during acute hepatitis C correlates with clinical outcomes. METHODS: Participants identified from the Australian Trial in Acute Hepatitis C and the Networks study were followed longitudinally from the time of diagnosis of acute hepatitis C. Peripheral blood mononuclear cells (PBMCs) and plasma were collected at and 2 time points after diagnosis. At each time point, TLR2, TLR4, and CD86 expression on peripheral blood monocytes, natural killer (NK) cells, and NK T cells was measured, as well as the response of PBMCs to stimulation with TLR ligands. Cytokine and chemokine levels were measured in stimulated PBMCs and plasma. RESULTS: We identified 20 participants with acute hepatitis C (10 with hepatitis C virus [HCV] monoinfection and 10 with HCV and human immunodeficiency virus coinfection). Eleven participants (55%) spontaneously cleared HCV. Acute hepatitis C and spontaneous clearance was associated with lower TLR4 expression on monocytes (P = .009) and NK cells (P = .029). Acute hepatitis C and spontaneous clearance was also associated with a reduced interferon γ response to TLR4 (P = .038) and TLR7/8 stimulation (P = .035), a reduced interleukin 6 response to TLR7/8 stimulation (P = .037), and reduced IFN-γ-inducible protein 10 (IP-10) response to TLR2 stimulation (P = .042). Lower plasma IP-10 levels were associated with spontaneous clearance (P = .001). CONCLUSIONS: These findings implicate TLR4 signaling as playing a critical role in the outcome of acute hepatitis C.
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Hepatitis C/inmunología , Leucocitos Mononucleares/inmunología , Transducción de Señal , Receptor Toll-Like 2/análisis , Receptor Toll-Like 4/análisis , Adulto , Australia , Antígeno B7-2/análisis , Citocinas/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/química , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
The majority of cystic fibrosis (CF) patients acquire chronic Pseudomonas aeruginosa lung infection, resulting in increased mortality and morbidity. The chronic P. aeruginosa lung infection is characterized by bacteria growing in biofilm surrounded by polymorphonuclear neutrophils (PMNs). However, the infection is not eradicated and the inflammatory response leads to gradual degradation of the lung tissue. In CF patients, a Th2-dominated adaptive immune response with a pronounced antibody response is correlated with poorer outcome. Dendritic cells (DCs) are crucial in bridging the innate immune system with the adaptive immune response. Once activated, the DCs deliver a set of signals to uncommitted T cells that induce development, such as expansion of regulatory T cells and polarization of Th1, Th2 or Th17 subsets. In this study, we characterized DCs in lungs and regional lymph nodes in BALB/c mice infected using intratracheal installation of P. aeruginosa embedded in seaweed alginate in the lungs. A significantly elevated concentration of DCs was detected earlier in the lungs than in the regional lymph nodes. To evaluate whether the chronic P. aeruginosa lung infection leads to activation of DCs, costimulatory molecules CD80 and CD86 were analyzed. During infection, the DCs showed significant elevation of CD80 and CD86 expression in both the lungs and the regional lymph nodes. Interestingly, the percentage of CD86-positive cells was significantly higher than the percentage of CD80-positive cells in the lymph nodes. In addition, cytokine production from Lipopolysaccharides (LPS)-stimulated DCs was analyzed demonstrating elevated production of IL-6, IL-10 and IL-12. However, production of IL-12 was suppressed earlier than IL-6 and IL-10. These results support that DCs are involved in skewing of the Th1/Th2 balance in CF and may be a possible treatment target.