Aurora A shines on early cell activation

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 20-01-2017La activación de las células T depende de la capacidad del receptor de células T (TCR) para reconocer antígenos específicos en el contacto del complejo mayor de histocompatibilidad (MHC) de las células presentadoras de antígeno (APC). La unión del TCR al MHC promueve la formación de la sinapsis inmunológica (IS). En este proceso, el TCR y las moléculas que se asocian a él se localizan en el área central de la región de contacto entre la célula T y la APC, en lo que se conoce como el complejo de activación supramolecular central (cSMAC). Las moléculas de adhesión se trasladan al complejo de activación supramolecular periférico (pSMAC). Algunas de las proteínas esenciales en este proceso son los miembros de la familia de proteínas Src (Lck y Fyn). La proteína quinasa Lck fosforila los motivos de activación de inmunorreceptores basados en tirosina del complejo TCR/CD3 permitiendo el reclutamiento de moléculas esenciales para la ruta de activación del TCR y la formación de la IS. La formación de la IS también induce cambios en el citoesqueleto de tubulina, incluyendo la translocación del centrosoma o el centro organizador de microtúbulos (MTOC), hacia la IS, acompañado por el aparato de Golgi, los cuerpos multivesiculares y las mitocondrias. Estos cambios facilitan la secreción polarizada de citoquinas y exosomas hacia la APC. La polarización del MTOC dirige el crecimiento activo de microtúbulos (MT), constituyendo el núcleo de una densa red de MT que regula el tráfico vesicular en la SI. La proteína Aurora A es una serina/treonina quinasa que desempeña un papel crítico en la dinámica del centrosoma y del huso mitótico durante la división celular. Durante la maduración del centrosoma, Aurora A promueve el ensamblaje de MT mediante el reclutamiento de factores nucleadores y estabilizadores de los mismos. Debido a su papel en el control de la dinámica de MT, postulamos que Aurora A podría ejercer un papel importante en la activación de células T durante la formación de la SI. Encontramos que Aurora A se activa tras la estimulación del TCR y se localiza en la IS durante el contacto celular. Por otro lado, tanto la inhibición farmacológica como la depleción génica de Aurora A en células T humanas o de ratón altera severamente la dinámica de MT así como el transporte a la IS de microvesículas que contienen CD3. El bloqueo de Aurora A impide la activación del complejo TCR/CD3, interrumpiendo la activación temprana de las células T así como la expresión de los genes CD69, CD25 y IL-2. La inhibición de Aurora A causa la deslocalización de tirosina quinasa Lck en la región de la SI y disminuye sus niveles de fosforilación, lo que indica que Aurora A es necesaria para mantener a Lck en su forma activa. Estos hallazgos demuestran que Aurora A es una molécula reguladora importante en la señalización temprana y del citoesqueleto de tubulina durante la activación de los linfocitos T.T cell activation depends on the ability of the T cell receptor (TCR) to recognize specific antigens presented in the context of the major histocompatibility complex (MHC) on the antigenpresenting cell (APC). The binding of the TCR to MHC promotes the formation of the immune synapse (IS). In this process, the TCR and its associated molecules localize to a central area of the T-cell-APC contact, the central supramolecular activating complex (cSMAC). Adhesion molecules relocate to the peripheral supramolecular activating complex (pSMAC). Essential proteins in this process are the Src family kinase members (Lck and Fyn). Lck phosphorylates the immunoreceptor tyrosine-based activation (ITAM) motifs of the TCR/CD3 complex leading to the recruitment of key molecules for the downstream signalling pathways and the IS formation. The formation of the IS also triggers changes in the tubulin cytoskeleton, including the translocation of the centrosome or microtubule (MT)-organizing centre (MTOC), to the IS, which is accompanied by the Golgi Apparatus, multivesicular bodies and mitochondria. These changes facilitate the polarized secretion of cytokines and exosomes toward the APC. MTOC polarization orchestrates active MT growth and forms the core of a dense MT network that regulates vesicular traffic at the IS. Aurora A is a serine/threonine kinase that plays a critical role in centrosome and spindle dynamics during mitosis. During centrosome maturation, Aurora A promotes MT assembly by recruiting nucleation and stabilization factors. Due to its role in controlling MT dynamics, we hypothesized that Aurora A may play a role in the activation of T lymphocytes during IS formation. We found that Aurora A is activated upon TCR stimulation and localizes at the IS during contact. Moreover, inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3-bearing vesicles at the IS. Specific targeting of Aurora A impairs activation of the TCR/CD3 complex, preventing early T cell activation and downstream expression of CD69, CD25 and IL-2. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings establish Aurora A as a major regulator of early signaling and the tubulin cytoskeleton during T cell activation

Aurora A drives early signalling and vesicle dynamics during T-cell activation

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3z-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.We thank S. Bartlett for English editing and critical reading of the manuscript, Dr A. Akhmanova for providing reagents, Maria Navarro for the her critical reading of the manuscript and scientific recommendations, Miguel Vicente-Manzanares for his critical reading of the manuscript, and Aitana Sanguino and Maria Jose Lopez for the technical support. We also thank the Confocal Microscopy \& Dynamic Imaging Unit (CNIC), Madrid, Spain. This study was supported by grants SAF2011-25834, SAF2014-55579-R and BIO2012-37926 from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid ERC-2011-AdG 294340-GENTRIS and ERC-2013-AdG 334763-NOVARIPP. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation, and the Pro-CNIC Foundation.S

Aurora a drives early signalling and vesicle dynamics during T-cell activation

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3¿-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.Spanish Ministry of Economy and Competitiveness, NDISNET-S2011/BMD-2332 from the Comunidad de Madrid ERC-2011-AdG 294340-GENTRIS and ERC-2013-AdG 334763-NOVARIPP. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation, and the Pro-CNIC FoundationPeer Reviewe

Aurora A controls CD8+ T cell cytotoxic activity and antiviral response

Aurora A is a serine/threonine kinase whose role in cell cycle progression and tumour generation has been widely studied. Recent work has revealed an unexpected function for Aurora A during CD4+ T cell activation and, also, in graft versus host disease development. However, it remains unknown whether Aurora A is involved in CD8+ T cell effector function and in cytotoxic T lymphocyte-mediated antiviral response. Here, we show that Aurora A chemical inhibition leads to an impairment of both the peptide-specific cytotoxicity and the degranulation activity of CD8+ T cells. This finding was similarly proven for both mice and human CD8+ CTL activity. As a result of Aurora A blockade, we detected a reduction in the expression induced by T cell activation of genes classically related to the effector function of cytotoxic T lymphocytes such as granzyme B or perforin1. Finally, we have found that Aurora A is necessary for CD8+ T cell-mediated antiviral response, in an in vivo model of vaccinia virus infection. Thus, we can conclude that Aurora A activity is, indeed, needed for the proper effector function of cytotoxic T lymphocytes and for their activity against viral threats.This study was supported by grants SAF2017/82886-R and BIO2012-37926 from the Spanish Ministry of Economy and Competitiveness, INFLAMUNE-CAMS2017/BMD-3671 from the Comunidad de Madrid and ERC-2011-AdG 294340-GENTRIS. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII) and also CIBER Cardiovascular. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and it is a Severo Ochoa Centre of Excellence (SEV-2015-0505).S

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection

Antibodies enhance the suppressive activity of extracellular vesicles in mouse delayed-type hypersensitivity

Previously, we showed that mouse delayed-type hypersensitivity (DTH) can be antigen-specifically downregulated by suppressor T cell-derived miRNA-150 carried by extracellular vesicles (EVs) that target antigen-presenting macrophages. However, the exact mechanism of the suppressive action of miRNA-150-targeted macrophages on effector T cells remained unclear, and our current studies aimed to investigate it. By employing the DTH mouse model, we showed that effector T cells were inhibited by macrophage-released EVs in a miRNA-150-dependent manner. This effect was enhanced by the pre-incubation of EVs with antigen-specific antibodies. Their specific binding to MHC class II-expressing EVs was proved in flow cytometry and ELISA-based experiments. Furthermore, by the use of nanoparticle tracking analysis and transmission electron microscopy, we found that the incubation of macrophage-released EVs with antigen-specific antibodies resulted in EVs’ aggregation, which significantly enhanced their suppressive activity in vivo. Nowadays, it is increasingly evident that EVs play an exceptional role in intercellular communication and selective cargo transfer, and thus are considered promising candidates for therapeutic usage. However, EVs appear to be less effective than their parental cells. In this context, our current studies provide evidence that antigen-specific antibodies can be easily used for increasing EVs’ biological activity, which has great therapeutic potential

<i>Hdac6</i>^<i>-/-</i> BMDCs show defective inflammatory cytokine response to Toll-like receptor stimuli.

<p>A) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with Pam3GSK4. ***p≤0.001, ** p≤0.01, * p≤0.05, ns>0.05 non-significant; n = 5–6. B) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with LPS. ***p≤0.001, ** p≤0.01, ns>0.05 non-significant; n = 5–6. C) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with Imiquimod. ***p≤0.001, * p≤0.05, ns>0.05 non-significant; n = 5–6. D) ELISA analysis of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL12p70 (pg/ml) in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs after treatment for 6, 12 and 24 h with HKST. ***p≤0.001, ** p≤0.01, ns>0.05 non-significant; n = 5–6.</p

<i>Hdac6</i>^<i>-/-</i> BMDCs accumulate higher levels of p62.

<p>A) <i>Left panels</i>: The charts show geometric means of p62 <i>and Lm</i> gated in the MHCII⁺CD11c⁺ population of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs without infection (NI) and at 6 hpi, with and without bafilomycin A1 treatment. The representative histograms on the right show p62 and <i>Lm</i> with and without bafilomycin A1. ***p≤0.001, ** p≤0.01, ns>0.05 non-significant; n = 6. B) Confocal microscopy analysis of p62-<i>Lm</i> co-localization in <i>Lm</i>-infected <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs at 6 hpi. Panels show DAPI (blue), <i>Lm</i> (red), p62 (green), and merged views of the three channels, with magnified views of the boxed areas. Yellow indicates p62-<i>Lm</i> co-localization. Scale bars 20 μm (main panels) and 10 μm (magnified views). <i>Right panel</i>: The chart shows ImarisCell Module analysis of the number of cells and the number of bacteria per cell in all pictures (10 pictures per genotype). Co-localization percentages were obtained by measuring the p62 channel on the bacterial surface using a threshold of 100. The statistical analysis of Imaris quantifications corresponds to the percentage of p62-<i>Lm</i> co-localization at 6 hpi. *p≤0.05; n = 10. C) Confocal microscopy analysis of actin-<i>Lm</i> co-localization in <i>Lm</i>-infected <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs at 6 hpi. Panels show DAPI (blue), <i>Lm</i> (green), β-actin (red), and merged views of the three channels, with magnified views of the boxed areas. Yellow indicates β-actin-<i>Lm</i> co-localization. Scale bars 20 μm (main panels) and 10 μm (magnified views). <i>Right panel</i>: The chart shows ImarisCell Module analysis of the number of cells and the number of bacteria per cell in all pictures (10 pictures per genotype). Co-localization percentages were obtained by measuring the actin channel on the bacterial surface using a threshold of 40.6. The statistical analysis of Imaris quantifications corresponds to the percentage of actin-<i>Lm</i> co-localization at 6 hpi. *** p≤0.001; n = 10. D) Confocal microscopy analysis of acetylated cortactin-<i>Lm</i> co-localization in <i>Lm</i>-infected <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs at 6 hpi. Panels show DAPI (blue), <i>Lm</i> (red), acetylated cortactin (green), and merged views of the three channels, with magnified views of the boxed areas. Yellow indicates acetylated cortactin-<i>Lm</i> co-localization. Scale bars 20 μm (main panels) and 10 μm (magnified views). <i>Right panel</i>: The chart shows ImarisCell Module analysis of the number of cells and the number of bacteria per cell in all pictures (10 pictures per genotype). Co-localization percentages were obtained by measuring the acetylated cortactin channel on the bacterial surface using a threshold of 184. The statistical analysis of Imaris quantifications corresponds to the percentage of acetylated cortactin-<i>Lm</i> co-localization at 6 hpi. *** p≤0.001; n = 10.</p

Dual role of HDAC6 during <i>Lm</i> infection in dendritic cells.

<p>The scheme shows the involvement of HDAC6 in two different functions of dendritic cell during <i>Lm</i> infection, the autophagy and the TLR signalling. <b>(1)</b> The fusion of phagosome with lysosome is dependent on cortactin and F-actin. The deacetylation of cortactin by HDAC6 allows the correct fusion, followed by an autophagic clearance of <i>Lm</i>. The absence of this enzyme delays the fusion of phagosome and lysosome, facilitating <i>Lm</i> to escape from phagosome leading to an increased bacterial load. <b>(2)</b> Di- and tri-acyl lipopeptides and peptidoglycan (PGN) of <i>Lm</i> are recognized by TLR1/2 or TLR2/6, activating the TLR pathway. HDAC6 is able to interact with the TLR-adaptor protein MyD88 which caused an enhanced down-stream signalling of TLR pathway, increasing NF-κB and MAPK activation. This stronger activation (independent on HDAC6 enzymatic activity) results in higher pro-inflammatory cytokine production and iNOS induction, reinforcing the ability of the DC to combat against this intracellular pathogen. Although the absence of HDAC6 does not fully abolish the activation of the DC, a lower induction of NF-κB and MAPK pathways promotes a lower activation of the anti-bacterial transcriptional program of the DCs. Note that both processes occur during <i>Lm</i> infection and the pro-inflammatory cytokines and iNOS induction can impact on the autophagic process. The images in the figure are not scaled.</p