Dataset Information

TLR3-/4-Priming Differentially Promotes Ca(2+) Signaling and Cytokine Expression and Ca(2+)-Dependently Augments Cytokine Release in hMSCs.

ABSTRACT: In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP3R) expression and IP3R-mediated Ca(2+) release, but also promoted Orai and STIM expression as well as store-operated Ca(2+) entry into hMSCs. In addition, we also observed that 21 Ca(2+) signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca(2+). These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca(2+) signaling and cytokine expression, and Ca(2+) -dependently potentiates cytokine release in hMSCs.

SUBMITTER: Park KS

PROVIDER: S-EPMC4793222 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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TLR3-/4-Priming Differentially Promotes Ca(2+) Signaling and Cytokine Expression and Ca(2+)-Dependently Augments Cytokine Release in hMSCs.

Park Kyoung Sun KS Kim Sun Hwa SH Das Amitabh A Yang Shao-Nian SN Jung Kyoung Hwa KH Kim Mi Kyung MK Berggren Per-Olof PO Lee YoungSeek Y Chai Jin Choul JC Kim Hyun Jin HJ Chai Young Gyu YG

Scientific reports 20160316

In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels o ...[more]

PMID: 26980664

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Project description:BackgroundMesenchymal stromal cells (MSCs) have regenerative and immunomodulatory properties, making them suitable for cell therapy. Toll-like receptors (TLRs) in MSCs respond to viral load by secreting immunosuppressive or proinflammatory molecules. The expression of anti-inflammatory molecules in MSCs can be altered by the concentration and duration of exposure to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)). This study aimed to optimize the preconditioning of MSCs with poly(I:C) to increase immunosuppressive effects and to identify MSCs with activated TLR3 (prMSCs).MethodsFlow cytometry and histochemical staining were used to analyze MSCs for immunophenotype and differentiation potential. MSCs were exposed to poly(I:C) at 1 and 10 μg/mL for 1, 3, and 24 h, followed by determination of the expression of IDO1, WARS1, PD-L1, TSG-6, and PTGES2 and PGE2 secretion. MSCs and prMSCs were cocultured with intact (J-) and activated (J+) Jurkat T cells. The proportion of proliferating and apoptotic J+ and J- cells, IL-10 secretion, and IL-2 production after cocultivation with MSCs and prMSCs were measured. Liquid chromatography-mass spectrometry and bioinformatics analysis identified proteins linked to TLR3 activation in MSCs.ResultsPoly(I:C) at 10 μg/mL during a 3-h incubation caused the highest expression of immunosuppression markers in MSCs. Activation of prMSCs caused a 18% decrease in proliferation and a one-third increase in apoptotic J+ cells compared to intact MSCs. Cocultures of prMSCs and Jurkat cells had increased IL-10 and decreased IL-2 in the conditioned medium. A proteomic study of MSCs and prMSCs identified 53 proteins with altered expression. Filtering the dataset with Gene Ontology and Reactome Pathway revealed that poly(I:C)-induced proteins activate the antiviral response. Protein‒protein interactions by String in prMSCs revealed that the antiviral response and IFN I signaling circuits were more active than in native MSCs. prMSCs expressed more cell adhesion proteins (ICAM-I and Galectin-3), PARP14, PSMB8, USP18, and GBP4, which may explain their anti-inflammatory effects on Jurkat cells.ConclusionsTLR3 activation in MSCs is dependent on exposure time and poly(I:C) concentration. The maximum expression of immunosuppressive molecules was observed with 10 µg/mL poly(I:C) for 3-h preconditioning. This priming protocol for MSCs enhances the immunosuppressive effects of prMSCs on T cells.

Project description:Occupational exposure to respirable crystalline silica (cSiO2) has been etiologically linked to human autoimmunity. Intranasal instillation with cSiO2 triggers profuse inflammation in the lung and onset of autoimmunity in lupus-prone mice; however, dietary supplementation with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) abrogates these responses. Inflammasome activation, IL-1 cytokine release, and death in alveolar macrophages following cSiO2 exposure are early and critical events that likely contribute to triggering premature autoimmune pathogenesis by this particle. Here we tested the hypothesis that DHA suppresses cSiO2-induced NLRP3 inflammasome activation, IL-1 cytokine release, and cell death in the macrophage. The model used was the murine macrophage RAW 264.7 cell line stably transfected with the inflammasome adapter protein ASC (RAW-ASC). Following priming with LPS, both the canonical activator nigericin and cSiO2 elicited robust inflammasome activation in RAW-ASC cells, as reflected by IL-1β release and caspase-1 activation. These responses were greatly diminished or absent in wild-type RAW cells. In contrast to IL-1β, cSiO2 induced IL-1α release in both RAW-ASC and to a lesser extent in RAW-WT cells after LPS priming. cSiO2-driven effects in RAW-ASC cells were confirmed in bone-marrow derived macrophages. Pre-incubating RAW-ASC cells with 10 and 25 μM DHA for 24 h enriched this fatty acid in the phospholipids by 15- and 25-fold, respectively, at the expense of oleic acid. DHA pre-incubation suppressed inflammasome activation and release of IL-1β and IL-1α by nigericin, cSiO2, and two other crystals - monosodium urate and alum. DHA's suppressive effects were linked to inhibition of LPS-induced Nlrp3, Il1b, and Il1a transcription, potentially through the activation of PPARγ. Finally, nigericin-induced death was inflammasome-dependent, indicative of pyroptosis, and could be inhibited by DHA pretreatment. In contrast, cSiO2-induced death was inflammasome-independent and not inhibited by DHA. Taken together, these findings indicate that DHA suppresses cSiO2-induced inflammasome activation and IL-1 cytokine release in macrophages by acting at the level of priming, but was not protective against cSiO2-induced cell death.

Project description:Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release - synaptotagmin-1 - slower Ca(2+)-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca(2+) sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca(2+)-dependent action: a Ca(2+)-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca(2+)-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca(2+)-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca(2+)-dependent fusion from the NRP. We conclude that the elusive 'alternative Ca(2+) sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+)-dependent properties of secretion without assuming parallel pools or sensors.

Project description:The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration.

Dataset Information

TLR3-/4-Priming Differentially Promotes Ca(2+) Signaling and Cytokine Expression and Ca(2+)-Dependently Augments Cytokine Release in hMSCs.

Publications

TLR3-/4-Priming Differentially Promotes Ca(2+) Signaling and Cytokine Expression and Ca(2+)-Dependently Augments Cytokine Release in hMSCs.

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