P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells

T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.Fil: Barrera Avalos, Carlos. Universidad de Santiago de Chile; ChileFil: Briceño, Pedro. Universidad de Chile; ChileFil: Valdés, Daniel. Universidad de Santiago de Chile; ChileFil: Imarai, Mónica. Universidad de Santiago de Chile; ChileFil: Leiva Salcedo, Elías. Universidad de Santiago de Chile; ChileFil: Rojo, Leonel E.. Universidad de Santiago de Chile; ChileFil: Milla, Luis A.. Universidad de Santiago de Chile; ChileFil: Huidobro Toro, Juan Pablo. Universidad de Santiago de Chile; ChileFil: Robles Planells, Claudia. Universidad de Santiago de Chile; ChileFil: Escobar, Alejandro. Universidad de Chile; ChileFil: Di Virgilio, Francesco. Università di Ferrara; ItaliaFil: Moron, Victor Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Sauma, Daniela. Universidad de Chile; ChileFil: Acuña Castillo, Claudio. Universidad de Santiago de Chile; Chil

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system

Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 phosphorylation and Ca2+ mobilisation

Volume-sensitive outwardly rectifying (VSOR) Cl- channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H2O2) in VSOR Cl- channel activation. The aim of this study was to determine the signalling pathways responsible for H2O2-induced VSOR Cl- channel activation. In rat hepatoma (HTC) cells, H2O2 elicited a transient increase in tyrosine phosphorylation of phospholipase Cγ1 (PLCγ1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H2O2 triggered an increase in cytosolic [Ca2+] that paralleled the time course of PLCγ1 phosphorylation. The H2O2-induced [Ca2+]i rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP3R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCγ1 activation and/or [Ca2+]i rise, abolished H2O2-induced VSOR Cl- currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCγ1, H2O2 did not induce activation of VSOR Cl- currents. All these H2O2-induced effects were independent of extracellular Ca2+. Our findings suggest that activation of PLCγ1 and subsequent Ca2+i mobilisation mediate H2O2-induced VSOR Cl- currents, indicating that H2O2 operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges.This work was supported by FONDAP-Fondecyt 15010006, Chile.Peer reviewe

Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.Fondecyt Fondo Nacional de Desarrollo Cientifico y Tecnologico 11140731 1141132 1160518 Programa de Atraccion e Insercion de Capital Humano Avanzado (PAI) Comision Nacional de Investigacion Cientifica y Tecnologica 79140059 Comision Nacional de Investigacion Cientifica y Tecnologica Financiamiento Basal para Centros Cientificos y Tecnologicos de Excelencia FB 0807 National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) R01 NS042225 Iniciativa Cientifica Milenio of the Ministry of Economy, Development and Tourism (Chile

Hydrogen peroxide removes TRPM4 current desensitization conferring increased vulnerability to necrotic cell death

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca 2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-D-glucamine and by knocking down TRPM4 with a shRNA

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca2+-activated non-selective cation channel TRPM4

Artículo de publicación ISITransient receptor potential melastatin-like 4 (TRPM4) is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P-2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.FONDECYT 11121239 3120041 National Institutes of Health NS42225 FONDAP 15010006 MECESUP UCH030

T-kininogen, a cystatin-like molecule, inhibits ERK-dependent lymphocyte proliferation

Plasma levels of kininogens increase with age in both rats and humans. Kininogens are inhibitors of cysteine proteinases, and filarial cysteine proteinase inhibitors (cystatins) reduce the proliferation of T cells. We evaluated whether T-kininogen (T-KG) might mimic this effect, and here we present data indicating that exposure of either rat splenocytes or Jurkat cells to purified T-KG results in inhibition of both ERK activation and [H-3]-thymidine incorporation, both basal and in response to ConA or PHA. Interestingly, T-KG did not impair [H-3]-thymidine incorporation in response to IL-2, which requires primarily the activation of the JNK and Jak/STAT pathways. These effects were neither the consequence of increased cell death, nor required the activity of kinin receptors. Furthermore, when T cell receptor proximal events were bypassed by the use of PMA plus Calcium ionophore, T-KG no longer inhibited ERK activation, suggesting that inhibition occurs upstream of these events, possibly at the level of membrane associated signal transduction molecules. We conclude that, like filarial cystatins, T-KG inhibits ERK-dependent T cell proliferation, and these observations suggest a possible role for T-KG in immunosenescence

In Vivo Antitumor Effect against Murine Cells of CT26 Colon Cancer and EL4 Lymphoma by Autologous Whole Tumor Dead Cells

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice’s immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

Artículo de publicación ISICellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.Fondecyt 11121239 11140064 1120240 1121078 1120126 NIH R01 NS042225 Conicyt Millennium Institute on Immunology and Immunotherapy P09-016-