Activación por quimioquinas de Janus quinasas y su papel en la migración y activación de linfocitos T por células presentadoras de antígeno

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-06-2016Las Janus quinasas son tirosina quinasas que tienen un papel clave en la señalización intracelular derivada de la activación de los receptores de citoquinas, pero también participan en la señalización desencadenada tras la activación de los receptores de quimioquinas. Las quimioquinas son proteínas de bajo peso molecular que actúan como factores quimioatrayentes y participan en multitud de procesos fisiológicos y patológicos, como por ejemplo, en la migración de los linfocitos T vírgenes a los ganglios linfáticos. Empleando linfocitos T vírgenes purificados en los que se redujo la expresión de las Janus quinasas JAK1 y JAK2, estudiamos la implicación de estas dos quinasas en la ruta de señalización desencadenada tras la activación de los receptores de quimioquinas CXCR4 y CCR7 por sus ligandos CXCL12 y CCL21 respectivamente. Los linfocitos T deficientes en JAK1 y JAK2 mostraron una menor capacidad de migración “in vitro” en repuesta a CXCL12 y CCL21. El uso de microscopía intravital nos permitió observar que “in vivo” también presentaban un defecto en su capacidad para llegar a los ganglios linfáticos (homing). JAK1 y JAK2 juegan un papel clave en la activación de las integrinas desde los receptores de quimioquinas, de hecho, en experimentos de adhesión sobre ICAM-1 y VCAM-1 se aprecia una disminución en la capacidad de adhesión de los linfocitos T deficientes en JAK1 y JAK2 en respuesta a CXCL12 y CCL21. Además, también participan en la polimerización de actina y en la activación de las proteínas ezrina, radixina y moesina (ERM) promovida por quimioquinas. Por otro lado, el receptor CXCR4 está presente en la sinapsis inmunológica, situándose en el anillo periférico, pSMAC. Su activación por CXCL12 unido a los glicosaminoglicanos sobre la célula dendrítica contribuye a la activación de las integrinas, y por lo tanto a estabilizar el conjugado entre la célula presentadora de antígeno y el linfocito T. En el mecanismo molecular implicado participan dos rutas complementarias, por un lado la proteína Gi y la posterior activación de RhoA, y por otro lado, las quinasas JAK1 y JAK2 que vía Vav-1 y RhoA también conectan CXCR4 con las integrinas. El bloqueo de dicha señalización o la reducción de la expresión de JAK1 y JAK2 provocan defectos en la estructura de la sinapsis, que se traducen en una menor capacidad de activación y proliferación del linfocito TJanus kinases are a very important tyrosine kinase family in the cytokine signaling pathway, which also participates in the chemokine signaling pathway. Chemokines are low weight molecular proteins which provide directional cues for cells and participate in multiple physiological and pathological processes such us naïve T cells homing. Using purified JAK1 and JAK2 deficient naïve T cells we studied the role of these kinases in the CXCR4 and CCR7 signaling pathway triggered by their ligands CXCL12 and CCL21, respectively. JAK1 and JAK2 deficient T cells showed a defect in migration towards CXCL12 and CCL21 “in vitro”. Moreover, a defect in homing was observed using two-photon microscopy. JAK1 and JAK2 play an essential role in integrin activation triggered by chemokine receptors. In fact, the adhesion capacity of JAK1 and JAK2 deficient T cells triggered by CXCL12 and CCL21 is diminished in adhesion experiments using plates coated with ICAM-1 and VCAM-1. Furthermore, JAK1 and JAK2 also participate in actin polymerization and ERM proteins activation promoted by chemokines. On the other hand, the CXCR4 receptor is present in the peripheral ring of the immunological synapse (pSMAC) and its activation by CXCL12 together with the glycosaminoglycans present on the dendritic cell surface contribute to integrins activation, hence, improving the binding between the antigen presenting cell and the T cell. This molecular mechanism involves two complementary pathways: the Gi protein pathway triggering the activation of RhoA; and JAK1 and JAK2 which connect CXCR4 with integrins via Vav-1 and RhoA. Either the blockage of this pathway or the knock-down of JAK1 and JAK2 cause defects in the structure of the immunological synapse leading to a deficiency in the activation and proliferation of the T cel

T Cell Migration in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints, associated with synovial hyperplasia and with bone and cartilage destruction. Although the primacy of T cell-related events early in the disease continues to be debated, there is strong evidence that autoantigen recognition by specific T cells is crucial to the pathophysiology of rheumatoid synovitis. In addition, T cells are key components of the immune cell infiltrate detected in the joints of RA patients. Initial analysis of the cytokines released into the synovial membrane showed an imbalance, with a predominance of proinflammatory mediators, indicating a deleterious effect of Th1 T cells. There is nonetheless evidence that Th17 cells also play an important role in RA. T cells migrate from the bloodstream to the synovial tissue via their interactions with the endothelial cells that line synovial postcapillary venules. At this stage, selectins, integrins, and chemokines have a central role in blood cell invasion of synovial tissue, and therefore in the intensity of the inflammatory response. In this review, we will focus on the mechanisms involved in T cell attraction to the joint, the proteins involved in their extravasation from blood vessels, and the signaling pathways activated. Knowledge of these processes will lead to a better understanding of the mechanism by which the systemic immune response causes local joint disorders and will help to provide a molecular basis for therapeutic strategies.This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2011-27370 and SAF2014-53416R),the RETICS Program (RD12/0009/009;RIER), and the Madrid Regional Government (S2010/BMD-2350;RAPHYME).Peer reviewe

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.This work was supported in part by grants from the Spanish Ministry of Science, Innovation and Universities (SAF 2017-82940-R) and the RETICS Program of the Instituto de salud Carlos III (RD12/0009/009 and RD16/0012/0006; RIER). LMM and JV are supported by the COMFUTURO program of the Fundación General CSIC

Chemokine Detection Using Receptors Immobilized on an SPR Sensor Surface

Chemokines and their receptors take part in many physiological and pathological processes, and their dysregulated expression is linked to chronic inflammatory and autoimmune diseases, immunodeficiencies, and cancer. The chemokine receptors, members of the G protein-coupled receptor family, are integral membrane proteins, with seven-transmembrane domains that bind the chemokines and transmit signals through GTP-binding proteins. Many assays used to study the structure, conformation, or activation mechanism of these receptors are based on ligand-binding measurement,as are techniques to detect new agonists and antagonists that modulate chemokine function. Such methods require labeling of the chemokine and/or its receptor, whichcan alter their binding characteristics. Surface plasmon resonance (SPR) is a powerful technique for analysis of the interaction between immobilized receptors and ligands in solution, in real time, and without labeling. SPR measurements nonetheless require expression and purification steps that can alter the conformation, stability, and function of the chemokine and/or the chemokine receptor. In this review, we focus on distinct methods to immobilize chemokine receptors on the surface of an optical biosensor. We expose the advantages and disadvantages of different protocols used and describe in detail the method to retain viral particles as receptor carriers that can be used for SPR determinations

Separating actin-dependent chemokine receptor nanoclustering from dimerization indicates a role for clustering in CXCR4 signaling and function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivoThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF 2014-53416-R, SAF 2017-82940-R AEI/FEDER, EU) and the RETICS Program (RD16/0012/0006; RIER), Ministry of Economy and Competitiveness, Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2013-0347; SEV-2015-0522), and Fundación Privada Cellex and Generalitat de Catalunya (CERCA program). L.M.-M. is supported by the COMFUTURO program of the Spanish Research Council General Foundation.Peer reviewe

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients

SignificanceNew imaging-based approaches are incorporating new concepts to our knowledge of biological processes. The analysis of receptor dynamics involved in cell movement using single-particle tracking demonstrates that cells require chemokine-mediated receptor clustering to sense appropriately chemoattractant gradients. Here, we report that this process does not occur in T cells expressing CXCR4R334X, a mutant form of CXCR4 linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). The underlaying molecular mechanism involves inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4R334X, which alters its lateral mobility and spatial organization. These defects, associated to CXCR4R334X expression, contribute to the retention of hematopoietic precursors in bone marrow niches and explain the severe immunological symptoms associated with WHIM syndrome.ISSN:0027-8424ISSN:1091-649