RESUMEN
Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Serina C-Palmitoiltransferasa/genética , Animales , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/inmunología , Femenino , Humanos , Coriomeningitis Linfocítica/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Transducción de Señal/inmunología , Esfingolípidos/biosíntesisRESUMEN
The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. ß2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated ß2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4+CD28null T cells represent a highly reactive subset showing 25-fold stronger ß2 integrin activation upon SDF-1α stimulation compared with CD28+ T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28null subset compared with the CD28+ T cells, whereas the established mediators of the classical pathways for ß2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28+ and CD28null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H2O2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization.
Asunto(s)
Síndrome Coronario Agudo/inmunología , Antígenos CD18/inmunología , Linfocitos T CD4-Positivos/inmunología , Señalización del Calcio/inmunología , Especies Reactivas de Oxígeno/inmunología , Síndrome Coronario Agudo/patología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/patología , Quimiocina CXCL12/inmunología , Femenino , Humanos , MasculinoRESUMEN
The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.
Asunto(s)
Actinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Linfocitos T/metabolismo , Células Cultivadas , Humanos , Fosforilación , Linfocitos T/inmunologíaRESUMEN
Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.
Asunto(s)
Fosfoproteínas Fosfatasas/inmunología , Transducción de Señal , Linfocitos T/inmunología , Biocatálisis , Diferenciación Celular , Células Cultivadas , Holoenzimas/inmunología , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Activación de Linfocitos , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfoproteínas Fosfatasas/genética , Interferencia de ARNRESUMEN
Cofilin is an essential actin remodeling protein promoting depolymerization and severing of actin filaments. To address the relevance of cofilin for the development and function of T cells in vivo, we generated knock-in mice in which T-cell-specific nonfunctional (nf) cofilin was expressed instead of wild-type (WT) cofilin. Nf cofilin mice lacked peripheral αß T cells and showed a severe thymus atrophy. This was caused by an early developmental arrest of thymocytes at the double negative (DN) stage. Importantly, even though DN thymocytes expressed the TCRß chain intracellularly, they completely lacked TCRß surface expression. In contrast, nf cofilin mice possessed normal numbers of γδ T cells. Their functionality was confirmed in the γδ T-cell-driven, imiquimod (IMQ)-induced, psoriasis-like murine model. Overall, this study not only highlights the importance of cofilin for early αß T-cell development but also shows for the first time that an actin-binding protein is differentially involved in αß versus γδ T-cell development.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Timo/metabolismo , Factores Despolimerizantes de la Actina/química , Animales , Movimiento Celular , Técnicas de Sustitución del Gen , Humanos , Células Jurkat , Ratones , Mutación/genética , Prolina/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Timocitos/metabolismoRESUMEN
The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell-cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.
Asunto(s)
Inmunomodulación , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Biomarcadores , Humanos , Vigilancia Inmunológica , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Neoplasias/metabolismo , Neoplasias/terapia , Infiltración Neutrófila , Especies Reactivas de Oxígeno , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T/metabolismo , Resultado del Tratamiento , Escape del Tumor/inmunología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patologíaRESUMEN
Osteoblasts lining the inner surface of bone support hematopoietic stem cell differentiation by virtue of proximity to the bone marrow. The osteoblasts also modify their own differentiation by producing various isoforms of fibronectin (FN). Despite evidence for immune regulation by osteoblasts, there is limited knowledge of how osteoblasts modulate cells of the immune system. Here, we show that extra domain A (EDA)-FN produced by osteoblasts increases arginase production in myeloid-derived cells, and we identify α5ß1 as the mediating receptor. In different mouse models of cancer, osteoblasts or EDA-FN was found to up-regulate arginase-1 expression in myeloid-derived cells, resulting in increased cancer growth. This harmful effect can be reduced by interfering with the integrin α5ß1 receptor or inhibiting arginase. Conversely, in tissue injury, the expression of arginase-1 is normally beneficial as it dampens the immune response to allow wound healing. We show that EDA-FN protects against excessive fibrotic tissue formation in a liver fibrosis model. Our results establish an immune regulatory function for EDA-FN originating from the osteoblasts and identify new avenues for enhancing the immune reaction against cancer.
RESUMEN
OBJECTIVE AND DESIGN: Abdominal aortic aneurysm (AAA) is heavily infiltrated with leukocytes, expressing the DNA sensor absent in melanoma 2 (AIM2) and other inflammasome components. METHODS: Using multicolour flow cytometry, we here compared the expression of the inflammasome components AIM2, NLRP3, and ASC in different peripheral immune cells derived from AAA patients with those from non-AAA patients in a case-control study. In parallel, peripheral blood mononuclear cells (PBMC) of AAA patients and controls were stimulated in vitro with poly-dA:dT or lipopolysaccharide (LPS) to analyze inflammasome activation. RESULTS: AIM2 expression was significantly increased in peripheral granulocytes (P = 0.026), monocytes (P = 0.007), B lymphocytes (P < 0.0001), and T lymphocytes (P = 0.004) of AAA patients. Expression of other inflammasome components did not differ between the groups. Following in vitro stimulation with foreign DNA, PBMC derived from AAA patients released significantly more IL-1ß (P = 0.022) into the supernatant than PBMC from control patients. In contrast, IL-1ß release upon LPS stimulation did not differ between the PBMC groups. CONCLUSION: The data indicate the increased activation of an AIM2 inflammasome in peripheral immune cells of AAA patients and point to a systemic AIM2-associated immune response to AAA.
Asunto(s)
Aneurisma de la Aorta Abdominal/inmunología , Proteínas de Unión al ADN/inmunología , Inflamasomas/inmunología , Leucocitos Mononucleares/inmunología , Anciano , ADN/inmunología , Femenino , Humanos , Interferón beta/sangre , Interleucina-1beta/sangre , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana EdadRESUMEN
NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.
Asunto(s)
Actinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Activación de Linfocitos , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Actinas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Bazo/inmunología , Bazo/patología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Linfocitos T/patología , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunología , Quinasa de Factor Nuclear kappa BRESUMEN
Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Comunicación Celular/inmunología , Citometría de Flujo/métodos , Citometría de Imagen/métodos , Microscopía/métodos , Linfocitos T/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/inmunología , Citoesqueleto de Actina/ultraestructura , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/ultraestructura , Antígenos CD/genética , Antígenos CD/inmunología , Biomarcadores/metabolismo , Complejo CD3/genética , Complejo CD3/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Comunicación Celular/genética , Transdiferenciación Celular , Técnicas de Cocultivo , Citometría de Flujo/instrumentación , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Expresión Génica , Granulocitos/inmunología , Granulocitos/metabolismo , Granulocitos/ultraestructura , Humanos , Citometría de Imagen/instrumentación , Sinapsis Inmunológicas/genética , Sinapsis Inmunológicas/ultraestructura , Separación Inmunomagnética/métodos , Microscopía/instrumentación , Cultivo Primario de Células , Linfocitos T/inmunología , Linfocitos T/ultraestructuraRESUMEN
The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citometría de Flujo/métodos , Inmunidad Celular , Linfocitos T CD8-positivos/citología , Línea Celular , HumanosRESUMEN
Oxidative stress leads to impaired T cell activation. A central integrator of T cell activation is the actin-remodelling protein cofilin. Cofilin is activated through dephosphorylation at Ser3. Activated cofilin enables actin dynamics through severing and depolymerization of F-actin. Binding of cofilin to actin is required for formation of the immune synapse and T cell activation. Here, we showed that oxidatively stressed human T cells were impaired in chemotaxis- and costimulation-induced F-actin modulation. Although cofilin was dephosphorylated, steady-state F-actin levels increased under oxidative stress conditions. Mass spectrometry revealed that cofilin itself was a target for oxidation. Cofilin oxidation induced formation of an intramolecular disulfide bridge and loss of its Ser3 phosphorylation. Importantly, dephosphorylated oxidized cofilin, although still able to bind to F-actin, did not mediate F-actin depolymerization. Impairing actin dynamics through oxidation of cofilin provides a molecular explanation for the T cell hyporesponsiveness caused by oxidative stress.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Activación de Linfocitos , Neutrófilos/inmunología , Estrés Oxidativo , Linfocitos T/inmunología , Factores Despolimerizantes de la Actina/química , Antígenos CD28/inmunología , Complejo CD3/inmunología , Quimiotaxis de Leucocito , Humanos , Peróxido de Hidrógeno/metabolismo , Quinasas Lim/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , Oxidación-Reducción , Fosforilación , Linfocitos T/metabolismoRESUMEN
Cofilin is an actin-binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T-cell activation and migration. The activity of cofilin is spatio-temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho-, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate. Costimulation via the T-cell receptor/CD3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF1α (stromal cell-derived factor 1α) induce Ras-dependent dephosphorylation of cofilin, which is important for immune synapse formation, T-cell activation, and T-cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5-bisphosphate-bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well-established three signals for T-cell activation, this microenvironmental control of cofilin delivers a modulating signal for T-cell-dependent immune reactions. This fourth modulating signal highly impacts both initial T-cell activation and the effector phase of T-cell-mediated immune responses.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Actinas/metabolismo , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Movimiento Celular/inmunología , Quimiocinas/metabolismo , Humanos , Inmunidad Celular/fisiología , Activación de Linfocitos/inmunología , Oxidación-Reducción , Fosfolípidos/metabolismoRESUMEN
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Linfocitos T/metabolismo , Factores Despolimerizantes de la Actina/química , Factores Despolimerizantes de la Actina/genética , Actinas/metabolismo , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Oxidación-Reducción , Fosfatidilinositol 4,5-Difosfato/química , Fosforilación , Polimerizacion , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Fosforilación , Neoplasias de la Próstata/patología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two-dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three-dimensional (3D)-extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T-cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM-kinase 1 activity, thereby allowing dephosphorylation of the F-actin-remodelling protein cofilin. Interference with this MEK-cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D-extracellular matrices, but not on 2D substrates. This MEK-cofilin module may have an important function in the tissue positioning of T cells during an immune response.
Asunto(s)
Movimiento Celular , Transducción de Señal , Linfocitos T/fisiología , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Células Cultivadas , Quimiotaxis , Técnicas de Silenciamiento del Gen , Humanos , Quinasas Lim/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.
Asunto(s)
Células Dendríticas/patología , Inmunidad Innata/fisiología , Fagocitos/fisiología , Yersiniosis/inmunología , Yersinia enterocolitica/inmunología , Traslado Adoptivo , Animales , Bacterias/inmunología , Separación Celular , Células Cultivadas , Femenino , Homeostasis/inmunología , Ratones , Ratones Transgénicos , Neutrófilos/trasplante , Fagocitos/inmunología , Regulación hacia Arriba/inmunología , Yersiniosis/patología , Yersiniosis/terapiaRESUMEN
Introduction: The use of Complementary and Integrative Medicine (CIM) is very popular among the general population in Germany. However, international studies show that nurses, physicians, and other health care professionals (HCPs) at hospitals often do not feel sufficiently informed about different CIM approaches. Moreover, they do not feel trained enough to counsel their patients appropriately. In the German-speaking context, particularly within university hospitals, research on this subject is scarce. Therefore, the aim of this explorative study was to evaluate attitudes, subjective knowledge, and needs regarding CIM among HCPs with direct patient interaction across all four university hospitals in the federal state of Baden-Württemberg, Germany (Tübingen, Ulm, Freiburg, Heidelberg). Methods: The multicenter, cross-sectional, anonymous full survey was conducted online using a self-developed, semi-structured, web-based questionnaire. Recruitment took place via all-inclusive e-mail distribution lists of all four university hospitals. Results: A total of n = 2,026 participants (response rate varied by location from about 5 to 14%) fully answered the questionnaire. Nurses constituted the largest professional group (n = 1,196; 59%), followed by physicians (n = 567; 28%), physiotherapists (n = 54), psychologists (n = 48), midwives (n = 37), and other professions (n = 124). More than two-thirds (71%, n = 1,437) of the participants were female and 14% (n = 286) reported additional training in CIM. The overall attitude toward CIM (10-point Likert scale, 10 = "very favorable") was clearly positive (M ± SD: 7.43 ± 2.33), with notable differences between professional groups: midwives (9.05 ± 1.18), physiotherapists (8.44 ± 1.74), and nurses (8.08 ± 1.95) expressed the highest support, whereas physicians (5.80 ± 2.39) the lowest. 42% of the participants incorporated CIM in patient care (from 33% of physicians to 86% of midwives). Overall, relaxation therapy (n = 1,951; 96%), external applications (n = 1,911; 94%), massage (n = 1,836; 91%), and meditation/mindfulness (n = 1,812; 89%) were rated as useful or rather useful for patients. The average self-assessed knowledge level about CIM was moderate (M ± SD: 5.83 ± 2.03). Most of the participants found CIM training at university hospitals important and saw research about CIM as one of the tasks of university hospitals. The participants expressed the highest interest in education for acupuncture/acupressure, relaxation therapies, and manual medicine. Discussion: This comprehensive survey of health care professionals (HCPs) at university hospitals in Germany reveals a clearly positive disposition toward CIM, aligning with findings from other hospital-based surveys and highlighting differences among professional groups. While most therapies deemed beneficial for patient care are supported by positive evidence, further research is required for others. Given the average self-reported knowledge of CIM, targeted education is essential to meet the needs of both HCPs and patients and to ensure the provision of evidence-based information on the risks and benefits of CIM.
RESUMEN
Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.
Asunto(s)
Antiinflamatorios , Citocinas , Flores , Activación de Linfocitos , Matricaria , Extractos Vegetales , Linfocitos T , Humanos , Extractos Vegetales/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Matricaria/química , Antiinflamatorios/farmacología , Citocinas/metabolismo , Flores/química , Activación de Linfocitos/efectos de los fármacos , Raíces de Plantas/química , Células Cultivadas , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacosRESUMEN
The amino acid arginine is fundamentally involved in the regulation of the immune response during infection, inflammatory diseases and tumor growth. Arginine deficiency (e.g. due to the myeloid cell enzyme arginase) inhibits proliferation and effector functions of activated T lymphocytes. Here, we studied intracellular mechanisms mediating this suppression of human T lymphocytes. Our proteomic analysis revealed an impaired dephosphorylation of the actin-binding protein cofilin upon T-cell activation in the absence of arginine. We show that this correlates with alteration of actin polymerization and impaired accumulation of CD2 and CD3 in the evolving immunological synapse in T cell-antigen presenting cells conjugates. In contrast, T-cell cytokine synthesis is differentially regulated in human T lymphocytes in the absence of arginine. While the production of certain cytokines (e.g. IFN-γ) is severely reduced, T lymphocytes produce other cytokines (e.g. IL-2) independent of extracellular arginine. MEK and PI3K activity are reciprocally regulated in association with impaired cofilin dephosphorylation. Finally, we show that impaired cofilin dephosphorylation is also detectable in human T cells activated in a granulocyte-dominated purulent micromilieu due to arginase-mediated arginine depletion. Our novel results identify cofilin as a potential regulator of human T-cell activation under conditions of inflammatory arginine deficiency.