The Chemerin/ChemR23 System Does Not Affect the Pro-Inflammatory Response of Mouse and Human Macrophages Ex Vivo

Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23−/− mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells

Caractérisation fonctionnelle du récepteur FPR3 et de son ligand peptidique F2L dans le développement de réponses inflammatoires physiologiques et pathologiques

Tous les êtres vivants présentent un arsenal de défenses contre les pathogènes, et la réponse inflammatoire constitue le processus initial de cette défense, qui s’achève par la réparation des tissus lésés. Paradoxalement, un processus inflammatoire prolongé est également associé à de nombreuses pathologies comme l’athérosclérose, l’asthme, les maladies auto-immunes mais aussi certains cancers. Le recrutement excessif de leucocytes au site de l’inflammation est un processus commun à ces pathologies. Dès lors, la compréhension et la maîtrise du phénomène complexe et finement orchestré de la migration sélective des populations leucocytaires, appelée chimiotactisme, sont des enjeux majeurs de la recherche médicale contemporaine. Les récepteurs aux peptides formylés bactériens et mitochondriaux (FPRs) forment la première famille de récepteurs chimiotactiques identifiée. Elle comprend trois membres, FPR1, 2 et 3, présentant un haut niveau de similitude et partageant certains de leurs multiples ligands. Le troisième membre de ce groupe, FPR3, reste actuellement le moins bien connu. Récemment, un agoniste de FPR3, affin et spécifique, a été identifié dans le laboratoire. Il s’agit du peptide F2L, qui correspond aux 21 premiers acides aminés de la protéine intracellulaire HEBP1.Dans le cadre de ce travail de thèse, nous nous sommes attelé à la caractérisation approfondie du récepteur FPR3 et son ligand peptidique F2L. Dans un premier temps, et à l’aide d’anticorps validés dans le cadre de ce travail, nous avons montré que le peptide F2L induit le chimiotactisme d’un ensemble de populations leucocytaires qui expriment FPR3, dont les sous-populations de macrophages des poumons, du colon et de la peau, les éosinophiles et les cellules dendritiques plasmacytoïdes. Cette distribution suggère, pour FPR3, une fonction dans la réponse inflammatoire. Nous avons pu montrer ensuite que F2L peut être généré par la protéolyse de son précurseur, HEBP1, sous l’action de la cathepsine D des macrophages. La cathepsine D est une aspartique protéase lysosomiale impliquée dans l’homéostasie cellulaire, les processus apoptotiques et inflammatoires physiologiques et pathologiques, et dans le développement tumoral. Il s’agit désormais d’identifier dans quel compartiment et sous quelles conditions F2L est produit et sécrété. Enfin, parallèlement à ces travaux, nous avons démontré que la cathepsine G, une sérine protéase contenue dans les granules azurophiles des neutrophiles, active également le récepteur FPR3. Des résultats préliminaires suggèrent un mode d’activation alternatif du récepteur, impliquant la protéolyse d’un troisième partenaire et la génération d’un agoniste actuellement non identifié. Le couple FPR3-F2L semble dès lors impliqué dans l’induction ou la résolution de la réponse inflammatoire en recrutant les éosinophiles, monocytes, macrophages et cellules dendritiques au site de la lésion.Doctorat en Sciences agronomiques et ingénierie biologiqueinfo:eu-repo/semantics/nonPublishe

Caractérisation fonctionnelle du récepteur FPR3 et de son ligand peptidique F2L dans le développement de réponses inflammatoires physiologiques et pathologiques

Tous les êtres vivants présentent un arsenal de défenses contre les pathogènes, et la réponse inflammatoire constitue le processus initial de cette défense, qui s’achève par la réparation des tissus lésés. Paradoxalement, un processus inflammatoire prolongé est également associé à de nombreuses pathologies comme l’athérosclérose, l’asthme, les maladies auto-immunes mais aussi certains cancers. Le recrutement excessif de leucocytes au site de l’inflammation est un processus commun à ces pathologies. Dès lors, la compréhension et la maîtrise du phénomène complexe et finement orchestré de la migration sélective des populations leucocytaires, appelée chimiotactisme, sont des enjeux majeurs de la recherche médicale contemporaine. Les récepteurs aux peptides formylés bactériens et mitochondriaux (FPRs) forment la première famille de récepteurs chimiotactiques identifiée. Elle comprend trois membres, FPR1, 2 et 3, présentant un haut niveau de similitude et partageant certains de leurs multiples ligands. Le troisième membre de ce groupe, FPR3, reste actuellement le moins bien connu. Récemment, un agoniste de FPR3, affin et spécifique, a été identifié dans le laboratoire. Il s’agit du peptide F2L, qui correspond aux 21 premiers acides aminés de la protéine intracellulaire HEBP1.Dans le cadre de ce travail de thèse, nous nous sommes attelé à la caractérisation approfondie du récepteur FPR3 et son ligand peptidique F2L. Dans un premier temps, et à l’aide d’anticorps validés dans le cadre de ce travail, nous avons montré que le peptide F2L induit le chimiotactisme d’un ensemble de populations leucocytaires qui expriment FPR3, dont les sous-populations de macrophages des poumons, du colon et de la peau, les éosinophiles et les cellules dendritiques plasmacytoïdes. Cette distribution suggère, pour FPR3, une fonction dans la réponse inflammatoire. Nous avons pu montrer ensuite que F2L peut être généré par la protéolyse de son précurseur, HEBP1, sous l’action de la cathepsine D des macrophages. La cathepsine D est une aspartique protéase lysosomiale impliquée dans l’homéostasie cellulaire, les processus apoptotiques et inflammatoires physiologiques et pathologiques, et dans le développement tumoral. Il s’agit désormais d’identifier dans quel compartiment et sous quelles conditions F2L est produit et sécrété. Enfin, parallèlement à ces travaux, nous avons démontré que la cathepsine G, une sérine protéase contenue dans les granules azurophiles des neutrophiles, active également le récepteur FPR3. Des résultats préliminaires suggèrent un mode d’activation alternatif du récepteur, impliquant la protéolyse d’un troisième partenaire et la génération d’un agoniste actuellement non identifié. Le couple FPR3-F2L semble dès lors impliqué dans l’induction ou la résolution de la réponse inflammatoire en recrutant les éosinophiles, monocytes, macrophages et cellules dendritiques au site de la lésion.Doctorat en Sciences agronomiques et ingénierie biologiqueinfo:eu-repo/semantics/nonPublishe

Rhophilin-2 is targeted to late-endosomal structures of the vesicular machinery in the presence of activated RhoB.

Rhophilin-2 or p76(RBE), a protein whose expression is induced by the cyclic AMP pathway in thyrocytes, contains several protein-protein interaction domains including HR-1, Bro1 and PDZ domains, and is a partner of RhoB in its GTP-bound form (Eur J Biochem, 269(24): 6241-9, 2002). We here define its subcellular localization and dissect the significance of its domains. By subcellular fractionation and colocalization experiments, rhophilin-2 is recruited to subcellular organelles by activated RhoB-GTP. As for its yeast homologue, Npi3/Bro1p, the Bro1 domain of rhophilin-2 is necessary to its recruitment to the vesicular structures, which are not labeled for EEA1 nor Lamp1, but well with the late endosome marker CD63.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Formyl peptide receptor-like 2 is expressed and functional in plasmacytoid dendritic cells, tissue-specific macrophage subpopulations, and eosinophils.

The formyl peptide receptor (FPR) is a key player in innate immunity and host defense mechanisms. In humans and other primates, a cluster of genes encodes two related receptors, FPR-like 1 and FPR-like 2 (FPRL1 and FPRL2). Despite their high sequence similarity, the three receptors respond to different sets of ligands and display a different expression pattern in leukocyte populations. Unlike FPR and FPRL1, FPRL2 is absent from neutrophils, and two endogenous peptide agonists, F2L and humanin, were recently described. In the present work, we investigated the detailed functional distribution of FPRL2 in leukocytes by quantitative PCR, flow cytometry, immunohistochemistry, and chemotaxis assays, with the aim of raising hypotheses regarding its potential functions in the human body. We describe that FPRL2 is highly expressed and functional in plasmacytoid dendritic cells and up-regulated upon their maturation. FPRL2 is also expressed in eosinophils, which are recruited but do not degranulate in response to F2L. FPRL2 is expressed and functional in macrophages differentiated from monocytes in vitro in different conditions. However, in vivo, only specific subsets of macrophages express the receptor, particularly in the lung, colon, and skin, three organs chronically exposed to pathogens and exogenous aggressions. This distribution and the demonstration of the production of the F2L peptide in mice underline the potential role of FPRL2 in innate immunity and possibly in immune regulation and allergic diseases.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3.

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Dose-response effects of chemerin on the production of cytokines by activated peritoneal macrophages.

<p>Peritoneal macrophages from WT (white bars) or ChemR23^−/− (black bars) mice, collected following i.p. Bio-Gel injection and selected by adherence, were tested for their production of pro-inflammatory (IL-6, IL-1β and TNF-α) and anti-inflammatory (IL-10) cytokines in response to stimulation by LPS and IFN-γ, in the presence or not of graded concentrations of recombinant chemerin (from 10⁻¹² to 10⁻⁶ M). After 15 h of culture, supernatants were collected and cytokine levels were determined by ELISA. Results are expressed as the percentage of the LPS/IFN-γ-induced levels (left panels) or as pg/ml (right panels) and represent the mean ± SEM of 3 to 4 independent experiments.</p

Effect of chemerin on the production of cytokines by activated mouse macrophages.

<p>Peritoneal macrophages from WT mice, collected after i.p. thioglycolate injection, were exposed or not for 1 h to 100 nM recombinant mouse chemerin. The cells were further stimulated by 100 ng/ml LPS. Unstimulated cells were used as controls. (A) At the indicated time points, cells were harvested for the determination of transcript levels for IL-6, TNF-α and IL-10, using quantitative RT-PCR. Results are expressed as mean ± SEM for 2 independent experiments performed in duplicate. (B) At the indicated time points, IL-6, TNF-α and IL-10 levels were determined in the supernatants by ELISA. Results are expressed as mean ± SEM for 3 independent experiments performed in duplicate.</p

Effects of chemerin on the production of cytokines by stimulated human macrophages.

<p>Human macrophages were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040043#s2" target="_blank">Material and Methods</a> section and were tested for their production of IL-6, TNF-α, IL-1β and IL-12p35 following stimulation by LPS, in the absence or presence of 100 nM recombinant human chemerin. At various time points, cells were collected and cytokine transcript levels were determined by quantitative RT-PCR. Results are expressed as fold increase over basal levels and represent the mean ± SEM of 3 independent experiments.</p

Mouse peritoneal macrophages express a functional ChemR23 receptor.

<p>(A) Peritoneal macrophages from WT or ChemR23^−/− mice, collected following i.p. Bio-Gel injection, were stimulated for 15 h by 100 ng/ml LPS or left unstimulated. Cells were harvested, and expression of ChemR23 was determined by quantitative RT-PCR. Data are expressed as mean ± SEM for 3 independent experiments performed in duplicate. (B) Peritoneal cells from WT or ChemR23^−/− mice, collected following i.p. Bio-Gel injection, were analysed by flow cytometry, directly or after selection by adherence to plates. Cells were stained for leukocyte markers (F4-80 and Gr1) and expression of ChemR23 on F4-80⁺ Gr1⁻ (Mφ) and F4-80⁻ Gr1⁺ (PMN) cells was evaluated using flow cytometry. Histograms represent the fluorescence observed following ChemR23 staining on peritoneal cells from WT (black histogram) or ChemR23^−/− (white histogram) mice, compared to the isotype-matched control (dotted line). One representative experiment out of 3 is shown. (C) Total peritoneal cells from WT and ChemR23^−/− mice, collected following i.p. Bio-Gel injection, were incubated with increasing concentrations of radiolabeled chemerin (•). Non-specific binding was determined in the presence of a 100-fold excess of unlabeled chemerin (*), and specific binding (□) was calculated as the difference. One representative experiment out of 3 is shown. (D) Peritoneal macrophages from WT or ChemR23^−/− mice, collected following i.p. Bio-Gel injection, were tested for their ability to migrate in response to recombinant mouse chemerin. In the left panel (mean ± SEM for 3 independent experiments performed in quadruplicate), the whole populations of cells recovered was used. In the right panel (mean ± SD for an experiment performed in triplicate), macrophages selected by overnight adherence were used. Results are expressed as migration index. (E) The biological activity of mouse recombinant chemerin was measured on mouse ChemR23-expressing CHO-K1 cells using the aequorin-based intracellular Ca²⁺ mobilization assay. Results are expressed as the percentage of the response to ATP and represent the mean ± SD of duplicated data points. One representative experiment out of 3 is shown.</p