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      CXCL14 Preferentially Synergizes With Homeostatic Chemokine Receptor Systems


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          Reflecting their importance in immunity, the activity of chemokines is regulated on several levels, including tissue and context-specific expression and availability of their cognate receptor on target cells. Chemokine synergism, affecting both chemokine and chemokine receptor function, has emerged as an additional control mechanism. We previously demonstrated that CXCL14 is a positive allosteric modulator of CXCR4 in its ability to synergize with CXCL12 in diverse cellular responses. Here, we have extended our study to additional homeostatic, as well as a selection of inflammatory chemokine systems. We report that CXCL14 strongly synergizes with low (sub-active) concentrations of CXCL13 and CCL19/CCL21 in in vitro chemotaxis with immune cells expressing the corresponding receptors CXCR5 and CCR7, respectively. CXCL14 by itself was inactive, not only on cells expressing CXCR5 or CCR7 but also on cells expressing any other known conventional or atypical chemokine receptor, as assessed by chemotaxis and/or β-arrestin recruitment assays. Furthermore, synergistic migration responses between CXCL14 and inflammatory chemokines CXCL10/CXCL11 and CCL5, targeting CXCR3 and CCR5, respectively, were marginal and occasional synergistic Ca 2+ flux responses were observed. CXCL14 bound to 300-19 cells and interfered with CCL19 binding to CCR7-expressing cells, suggesting that these cellular interactions contributed to the reported CXCL14-mediated synergistic activities. We propose a model whereby tissue-expressed CXCL14 contributes to cell localization under steady-state conditions at sites with prominent expression of homeostatic chemokines.

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          Chemokines and chemokine receptors: positioning cells for host defense and immunity.

          Chemokines are chemotactic cytokines that control the migratory patterns and positioning of all immune cells. Although chemokines were initially appreciated as important mediators of acute inflammation, we now know that this complex system of approximately 50 endogenous chemokine ligands and 20 G protein-coupled seven-transmembrane signaling receptors is also critical for the generation of primary and secondary adaptive cellular and humoral immune responses. Recent studies demonstrate important roles for the chemokine system in the priming of naive T cells, in cell fate decisions such as effector and memory cell differentiation, and in regulatory T cell function. In this review, we focus on recent advances in understanding how the chemokine system orchestrates immune cell migration and positioning at the organismic level in homeostasis, in acute inflammation, and during the generation and regulation of adoptive primary and secondary immune responses in the lymphoid system and peripheral nonlymphoid tissue.
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            A new generation of Ca2+ indicators with greatly improved fluorescence properties.

            A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
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              NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.

              Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

                Author and article information

                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                05 October 2020
                : 11
                : 561404
                [1] 1Division of Infection and Immunity, Cardiff University School of Medicine , Cardiff, United Kingdom
                [2] 2Institute of Immunology and Immunotherapy, University of Birmingham , Birmingham, United Kingdom
                [3] 3Laboratory of Molecular Immunology, Rega Institute for Medical Research, KU Leuven , Leuven, Belgium
                [4] 4Department of Infection and Immunity, Immuno-Pharmacology and Interactomics, Luxembourg Institute of Health (LIH) , Esch-sur-Alzette, Luxembourg
                [5] 5Biotechnology Institute Thurgau (BITg), University of Konstanz , Kreuzlingen, Switzerland
                Author notes

                Edited by: Joanna Cichy, Jagiellonian University, Poland

                Reviewed by: Robert J. B. Nibbs, University of Glasgow, United Kingdom; Irina Kufareva, University of California, San Diego, United States

                *Correspondence: Bernhard Moser, moserb@ 123456cardiff.ac.uk

                These authors have contributed equally to this work

                This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology

                Copyright © 2020 Kouzeli, Collins, Metzemaekers, Meyrath, Szpakowska, Artinger, Struyf, Proost, Chevigne, Legler, Eberl and Moser.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                : 12 May 2020
                : 02 September 2020
                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 66, Pages: 14, Words: 0
                Funded by: Medical Research Council 10.13039/501100000265
                Funded by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung 10.13039/501100001711
                Funded by: KU Leuven 10.13039/501100004040
                Funded by: Fonds National de la Recherche Luxembourg 10.13039/501100001866
                Original Research

                chemokines,signal transduction,synergism,migration,cell localization,cxcr4,cxcr5, ccr7
                chemokines, signal transduction, synergism, migration, cell localization, cxcr4, cxcr5, ccr7


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