RESUMEN

Recently, aging is becoming an important social problem in many developed countries including Japan. It is socially and universally important to unveil the impact of aging and extend healthy life expectancy. Here we show our recent finding that dedicator of cytokinesis 11 (DOCK11, also known as Zizimin2) may be involved in immunosenescence of B cells. DOCK11 was identified as a guanine nucleotide exchange factor for a small GTPase called cell division cycle 42. Expression of DOCK11 is restricted to lymphoid tissues, and becomes downregulated with age. Thus we examined the involvement of DOCK11 in immunosenescence of B-1a B cells as an example. B-1a cells are the main source of antibodies at steady state, and function as the first line of defense against infection. Although DOCK11 was expressed by B-1a cells, the expression levels declined with age. Furthermore, production of anti-pneumococcal immunoglobulin M antibodies was suppressed in aged mice, and was recovered by adoptive transfer with B-1a cells in a DOCK11-dependent manner. Thus DOCK11 may be involved in immunosenescence of B-1a cells.

Asunto(s)

Envejecimiento/inmunología , Inmunosenescencia , Animales , Linfocitos B/inmunología , Citocinesis/inmunología , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Inmunoglobulina M , Ratones , Estado Nutricional , Streptococcus pneumoniae/inmunología

RESUMEN

Connections established between cytoskeleton and plasma membrane are essential in cellular processes such as cell migration, vesicular trafficking, and cytokinesis. Class I myosins are motor proteins linking the actin-cytoskeleton with membrane phospholipids. Previous studies have implicated these molecules in cell functions including endocytosis, exocytosis, release of extracellular vesicles and the regulation of cell shape and membrane elasticity. In immune cells, those proteins also are involved in the formation and maintenance of immunological synapse-related signaling. Thus, these proteins are master regulators of actin cytoskeleton dynamics in different scenarios. Although the localization of class I myosins has been described in vertebrates, their functions, regulation, and mechanical properties are not very well understood. In this review, we focused on and summarized the current understanding of class I myosins in vertebrates with particular emphasis in leukocytes.

Asunto(s)

Citoesqueleto de Actina/inmunología , Sistema Inmunológico/citología , Sinapsis Inmunológicas/metabolismo , Leucocitos/inmunología , Miosina Tipo I/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Movimiento Celular , Citocinesis/genética , Citocinesis/inmunología , Endocitosis , Exocitosis , Vesículas Extracelulares/química , Vesículas Extracelulares/inmunología , Regulación de la Expresión Génica , Humanos , Leucocitos/metabolismo , Leucocitos/ultraestructura , Mecanotransducción Celular/inmunología , Miosina Tipo I/clasificación , Miosina Tipo I/inmunología , Fosfolípidos/inmunología , Fosfolípidos/metabolismo , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología

RESUMEN

There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.

Asunto(s)

Citocinesis , Leishmania infantum , Leishmaniasis Visceral , Mutación , Animales , Citocinesis/genética , Citocinesis/inmunología , Modelos Animales de Enfermedad , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/inmunología , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmania infantum/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/prevención & control , Puntos de Control de la Fase M del Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Plásmidos/genética , Plásmidos/inmunología , Plásmidos/metabolismo , Células THP-1

RESUMEN

Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving. Here we describe the methods currently applied to monitor and measure ACD of developing and activated T cells. We provide an overview of approaches for capturing cells in the process of cytokinesis in vivo, ex vivo, or during in vitro culture. We provide methods for in vitro fixed immunofluorescent staining and for time-lapse analysis. We provide an overview of the different approaches for quantification of ACD of lymphocytes, discuss the pitfalls and concerns in interpretation of these analyses, and provide detailed methods for the quantification of ACD in our group.

Asunto(s)

División Celular Asimétrica/inmunología , Caenorhabditis elegans/inmunología , Citocinesis/inmunología , Linfocitos T/inmunología , Animales , Drosophila melanogaster , Microscopía Fluorescente/métodos , Linfocitos T/citología

RESUMEN

T cell proliferation is initiated by T cell antigen receptor (TCR) triggering, soluble growth factors or both. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and septin-independent pathways. Septin-deficient T cells failed to complete cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was not dependent on septins when cell-cell contacts, such as those with antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and costimulatory signals. We were able to differentiate between cytokine- and antigen-driven expansion in vivo and thus show that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Proliferación Celular/genética , Citocinesis/inmunología , Septinas/inmunología , Animales , Células Presentadoras de Antígenos , Señalización del Calcio , Citocinas/farmacología , Citocinesis/efectos de los fármacos , Citocinesis/genética , Citometría de Flujo , Immunoblotting , Integrinas , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Fosfatidilinositol 3-Quinasas , Fosforilación , Receptores de Antígenos de Linfocitos T , Factor de Transcripción STAT5/metabolismo , Septinas/genética

RESUMEN

Tissues accommodate defined numbers of dendritic cells (DCs) in highly specific niches where different intrinsic and environmental stimuli control DC life span and numbers. DC homeostasis in tissues is important, because experimental changes in DC numbers influence immunity and tolerance toward various immune catastrophes and inflammation. However, the precise molecular mechanisms regulating DC life span and homeostasis are unclear. We report that the GTPase RhoA controls homeostatic proliferation, cytokinesis, survival, and turnover of cDCs. Deletion of RhoA strongly decreased the numbers of CD11b(-)CD8(+) and CD11b(+)Esam(hi) DC subsets, whereas CD11b(+)Esam(lo) DCs were not affected in conditional RhoA-deficient mice. Proteome analyses revealed a defective prosurvival pathway via PI3K/protein kinase B (Akt1)/Bcl-2-associated death promoter in the absence of RhoA. Taken together, our findings identify RhoA as a central regulator of DC homeostasis, and its deletion decreases DC numbers below critical thresholds for immune protection and homeostasis, causing aberrant compensatory DC proliferation.

Asunto(s)

Apoptosis/inmunología , Células Dendríticas/inmunología , Homeostasis/inmunología , Proteína de Unión al GTP rhoA/inmunología , Animales , Apoptosis/genética , Western Blotting , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Antígeno CD11c/inmunología , Antígeno CD11c/metabolismo , Proliferación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Citocinesis/genética , Citocinesis/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Homeostasis/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Proteína Letal Asociada a bcl/inmunología , Proteína Letal Asociada a bcl/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo

RESUMEN

Accumulating evidence underscores the immune synapse (IS) of naive T cells as a site of intense vesicular trafficking. At variance with helper and cytolytic effectors, which use the IS as a secretory platform to deliver cytokines and/or lytic granules to their cellular targets, this process is exploited by naive T cells as a means to regulate the assembly and maintenance of the IS, on which productive signaling and cell activation crucially depend. We have recently identified a role of the intraflagellar transport (IFT) system, which is responsible for the assembly of the primary cilium, in the non-ciliated T-cell, where it controls IS assembly by promoting polarized T-cell receptor recycling. This unexpected finding not only provides new insight into the mechanisms of IS assembly but also strongly supports the notion that the IS and the primary cilium, which are both characterized by a specialized membrane domain highly enriched in receptors and signaling mediators, share architectural similarities and are homologous structures. Here, we review our current understanding of vesicular trafficking in the regulation of the assembly and maintenance of the naive T-cell IS and the primary cilium, with a focus on the IFT system.

Asunto(s)

Compartimento Celular/inmunología , Cilios/inmunología , Sinapsis Inmunológicas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Vesículas Transportadoras/inmunología , Animales , Citocinas/inmunología , Citocinesis/inmunología , Citotoxicidad Inmunológica , Humanos , Transducción de Señal/inmunología

RESUMEN

The immune system is made up of a diverse collection of cells, each of which has distinct sets of triggers that elicit unique and overlapping responses. It is correctly described as a 'system' because its overall properties (e.g. 'tolerance', 'allergy') emerge from multiple interactions of its components cells. To mobilize a response where needed, the majority of the cells of the system are obligatorily highly motile and so must communicate with one another over both time and space. Here, we discuss the flexibility of the primary immunological synapse (IS) with respect to motility. We then consider the primary IS as an initiating module that licenses 'immunological circuits': the latter consisting of two or more cell-cell synaptic interactions. We discuss how two or three component immunological circuits interact might with one another in sequence and how the timing, stoichiometry, milieu, and duration of assembly of immunological circuits are likely to be key determinants in the emergent outcome and thus the system-wide immune response. An evolving consideration of immunological circuits, with an emphasis on the cell-cell modules that complement T-antigen-presenting cell interaction, provides a fundamental starting point for systems analysis of the immune response.

Asunto(s)

Comunicación Celular , Sistema Inmunológico , Inmunidad Celular , Sinapsis Inmunológicas/inmunología , Animales , Comunicación Celular/inmunología , Movimiento Celular/inmunología , Microambiente Celular/inmunología , Citocinesis/inmunología , Humanos , Receptor Cross-Talk , Transducción de Señal

RESUMEN

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.

Asunto(s)

Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Proliferación Celular , Citocinesis/inmunología , Centro Germinal/citología , Centro Germinal/inmunología , Modelos Inmunológicos , Animales , Adhesión Celular/inmunología , Agregación Celular/inmunología , Diferenciación Celular/inmunología , Estudios Transversales , Haptenos/administración & dosificación , Haptenos/inmunología , Ratones , Ratones Endogámicos BALB C , Oxazolona/administración & dosificación , Oxazolona/análogos & derivados , Oxazolona/inmunología , Bazo/citología , Bazo/inmunología , Procesos Estocásticos

RESUMEN

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2(+/+) mice and CCR2(-/-) mice throughout a time course following infection with C. neoformans. Results demonstrate that numbers of Ly-6C(high) monocytes increased in parallel in the peripheral blood and lungs of CCR(+/+) mice, whereas CD11c(+) MHC class II(+) pre-DC were 10-fold less prevalent in the peripheral blood and did not differ between the two strains. Accumulation of Ly-6C(high) monocytes correlated with a substantial increase in the numbers of CD11b(+) DC in the lungs of infected CCR2(+/+) mice. Comparative phenotypic analysis of lung cells recovered in vivo suggests that Ly-6C(high) monocytes differentiate into CD11b(+) DC in the lung; differentiation is associated with up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that Ly-6C(high) monocytes differentiate into CD11b(+) DC. Accumulation of Ly-6C(high) monocytes and CD11b(+) DC was not attributable to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11b(+) DC in the lungs of Cryptococcus-infected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6C(high) monocytes.

Asunto(s)

Antígenos Ly/biosíntesis , Antígeno CD11b/biosíntesis , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Enfermedades Pulmonares Fúngicas/inmunología , Pulmón/inmunología , Monocitos/inmunología , Receptores CCR2/fisiología , Animales , Antígenos Ly/fisiología , Recuento de Células , Diferenciación Celular/genética , Movimiento Celular/genética , Proliferación Celular , Criptococosis/inmunología , Criptococosis/metabolismo , Criptococosis/patología , Cryptococcus neoformans/inmunología , Citocinesis/genética , Citocinesis/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Femenino , Pulmón/metabolismo , Pulmón/patología , Enfermedades Pulmonares Fúngicas/microbiología , Enfermedades Pulmonares Fúngicas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patología , Receptores CCR2/biosíntesis , Receptores CCR2/deficiencia , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patología

RESUMEN

Our current view of T cell differentiation and population dynamics is assembled from pieces of data obtained from separate experimental systems and is thus patchy. We reassessed homeostasis and dynamics of T cells 1) by generating a mathematical model describing the spatiotemporal features of T cell differentiation, and 2) by fitting this model to experimental data generated by disturbing T cell differentiation through transient depletion of dividing T cells in mice. This specific depletion was obtained by administration of ganciclovir to mice expressing the conditional thymidine kinase suicide gene in T cells. With this experimental approach, we could derive quantitative parameters describing the cell fluxes, residence times, and rates of import, export, proliferation, and death across cell compartments for thymocytes and recent thymic emigrants (RTEs). Among other parameters, we show that 93% of thymocytes produced before single-positive stages are eliminated through the selection process. Then, a postselection peripheral expansion of naive T cells contributes three times more to naive T cell production than the thymus, with half of the naive T cells consisting of dividing RTEs. Altogether, this work provides a quantitative population dynamical framework of thymocyte development, RTEs, and naive T cells.

Asunto(s)

Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Homeostasis/inmunología , Modelos Inmunológicos , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Diferenciación Celular/genética , División Celular/genética , División Celular/inmunología , Movimiento Celular/genética , Citocinesis/genética , Citocinesis/inmunología , Ganciclovir/administración & dosificación , Herpesvirus Humano 1/genética , Homeostasis/genética , Recuento de Linfocitos , Depleción Linfocítica , Ratones , Ratones Transgénicos , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/inmunología , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/enzimología , Timidina Quinasa/biosíntesis , Timidina Quinasa/deficiencia , Timidina Quinasa/genética , Timo/citología , Timo/inmunología

RESUMEN

O objetivo dessa tese foi avaliar a expressão de citocinas Th1 (IL-12 e INFγ), citocinas Th2 (IL-4, IL-6 e IL-10) e das citocinas pró-inflamatórias IL-18, IL-1β e TNFα no fluido gengival de pacientes com periodontite crônica portadores da doença de Crohn (DC), de retocolite ulcerativa idiopática (RCUI) e em indivíduos saudáveis (o grupo controle, GC). Como objetivo secundário, avaliamos a função dos neutrófilos no fluido gengival desses pacientes através da mensuração das metaloproteinases da matriz -8, -9 (MMP-8 e MMP-9) e da atividade da elastase. Quinze pacientes com DC (idade média 38.2 ± 11.4 anos), 15 pacientes com RCUI (idade média 45.0 ± 10.5 anos) e 15 pacientes saudáveis (idade média 42.1 ± 7.8 anos) participaram desse estudo. Todos os dentes presentes, com exceção dos terceiros molares, foram examinados. Profundidade de bolsa (PB), nível de inserção clínica (NI), presença de placa e de sangramento a sondagem foram avaliados em seis sítios por dente. Em cada paciente, o fluido de 4 sítios com periodontite (PB ≥ 5 mm e NI ≥ 3mm) e de 4 sítios com gengivite (PB ≤ 3 mm e NI ≤ 1 mm) foram coletados através de pontas de papel absorvente pré-fabricadas. O sistema LUMINEX® foi utilizado na mensuração das IL-1β, IL-4, IL-6, IL-10, IL-12p70, TNFα, INFγ, MMP-8 e MMP-9. A IL-18 foi analisada através do ensaio ELISA e a atividade de elastase através de uma reação enzimática. O soro desses pacientes também foi analisado e o coeficiente de correlação de Pearson foi utilizado na análise da correlação entre as citocinas no soro e no fluido gengival. Nos sítios com gengivite, a quantidade total de IL-4 foi significativamente menor no grupo RCUI do que no grupo GC (p=0.016). Nos sítios com periodontite, a quantidade total de IL-4 foi significativamente menor no grupo DC do que no grupo GC (p=0.029)...

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The aim of this thesis was to evaluate the expression of Th1 cytokines (IL-12 and INF-γ), Th2 cytokines (IL-4, IL-6 and IL-10) and the pro-inflammatory cytokines IL-18, IL-1β and TNF-α in the gingival crevicular fluid (GCF) from Crohn’s disease (CD) patients, ulcerative colitis (UC) patients and healthy individuals (control group, CG) who had chronic periodontitis. Besides, we measured elastase activity, matrix metalloproteinase -8 and -9 (MMP-8 and -9) to address the neutrophil function in the GCF. Fifteen CD patients (mean age 38.2 ± 11.4 years), 15 UC patients (mean age 45.0 ± 10.5 years) and 15 systemically healthy controls (mean age 42.1 ± 7.8 years) were enrolled in this study. All the present teeth, except for the third molars were examined. Probing pocket depth (PPD), clinical attachment loss (CAL), presence of plaque and presence of bleeding on probing were assessed in six sites per tooth. In every subject, GCF from 4 gingivitis sites (PPD ≤ 3mm and CAL ≤ 1mm) and from 4 periodontitis sites (PPD ≥ 5mm and CAL ≥ 3mm) were collected with filter strips. The data were reported as total amount and concentration. IL-1β, IL-4, IL-6, IL-10, IL-12p70, TNFα, INFγ, MMP-8 and MMP-9 were analyzed by the Luminex® analyzer. IL-18 was analyzed using a commercially available ELISA assay and the elastase activity by an enzymatic reaction. The serum was also analysed and the correlations between the cytokines in the GCF and in the serum were calculated by Pearson correlation analysis. In gingivitis sites, the total amount of IL-4 was significantly lower in the UC group than in the CG group (p=0.016). In periodontitis sites, the total amount of IL-4 was significantly lower in CD group than in the CG group (p=0.029). The total amount of IL-4 was lower in UC group than in CD group (p=0.077)...

Asunto(s)

Humanos , Citocinas/química , Citocinesis/inmunología , Líquido del Surco Gingival/química , Linfocitos/química , Periodontitis Crónica/enzimología , Estudios de Casos y Controles , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Elastasa de Leucocito , Metaloproteinasas de la Matriz/química , Proctocolitis