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

ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model.

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis

International audienceChemerin is a multifunctional protein acting mainly through the G protein-coupled receptor ChemR23/CMKLR1/Chemerin1. Its expression is frequently downregulated in human tumors, including in melanoma and squamous cell carcinoma of the skin and anti-tumoral properties of chemerin were reported in mouse tumor graft models. In the present study, we report the development of spontaneous skin tumors in aged ChemR23-deficient mice. In order to test the potential therapeutic benefit of chemerin analogs, a transgenic model in which bioactive chemerin is over-expressed by basal keratinocytes was generated. These animals are characterized by increased levels of chemerin immunoreactivity and bioactivity in the skin and the circulation. In a chemical carcinogenesis model, papillomas developed later, were less numerous, and their progression to carcinomas was delayed. Temporal control of chemerin expression by doxycycline allowed to attribute its effects to late stages of carcinogenesis. The protective effects of chemerin were partly abrogated by ChemR23 invalidation. These results demonstrate that chemerin is able to delay very significantly tumor progression in a model that recapitulates closely the evolution of solid cancer types in human and suggest that the chemerin-ChemR23 system might constitute an interesting target for therapeutic intervention in the cancer field

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

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