Natural killer cells induce eosinophil activation and apoptosis.

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis

Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+ T-Cell Activation.

The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 upon in vivo activation of transformed and primary human CD4(+) T cells. We show that following activation of CD4(+) T cells, BCL11B still binds to IL-2 and Id2 promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression of BCL11B by KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4(+) T cells

Effect of diesel on chemokines and chemokine receptors involved in helper T cell type 1/type 2 recruitment in patients with asthma.

The objective of this study was to evaluate if diesel exhausts could favor helper T cell type (Th) 2-associated allergic reactions either through an increased production of Th2-associated chemokines and of their associated receptors or through a decrease of Th1-attracting chemokines and chemokine receptors. Diesel but not allergen exposure of peripheral blood mononuclear cells from subjects with allergy induced a release of I-309, whereas both diesel and Der p 1 induced an early but transient release of monokine induced by IFN-gamma and a late release of pulmonary and activation-regulated chemokine. Although both Th1- and Th2-attracting chemokines were induced, the resulting effect was an increased chemotactic activity on Th2 but not Th1 cells. Surprisingly, diesel induced a late increase in the expression of the Th1-associated CXC receptor 3 and CC receptor 5. T cell CXC receptor 3 upregulation was not associated with an increased migration to its ligands. These two antagonistic effects have been previously reported as a scavenger mechanism to clear chemokines. Altogether, these results suggest that diesel, even without allergen, may amplify a type 2 immune response but that it can also increase late Th1-associated chemokine receptor expression, perhaps as a scavenger mechanism to clear pro-Th1 chemokines and promote the Th2 pathway.In VitroJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Natural Killer Cells from Allergic Donors Are Defective in Their Response to CCL18 Chemokine

International audienceNatural killer (NK) cells were originally described as cytolytic effector cells, but since then have been recognized to possess regulatory functions on immune responses. Chemokines locate NK cells throughout the body in homeostatic and pathological conditions. They may also directly stimulate immune cells. CCL18 is a constitutive and inducible chemokine involved in allergic diseases. The aim of this study was to evaluate CCL18’s effect on NK cells from allergic and nonallergic donors in terms of both chemotactic and immune effects. Results showed that CCL18 was able to induce migration of NK cells from nonallergic donors in a G-protein-dependent manner, suggesting the involvement of a classical chemokine receptor from the family of seven-transmembrane domain G-protein-coupled receptors. In contrast, NK cells from allergic patients were unresponsive. Similarly, CCL18 was able to induce NK cell cytotoxicity only in nonallergic subjects. Purified NK cells did not express CCR8, one of the receptors described to be involved in CCL18 functions. Finally, the defect in CCL18 response by NK cells from allergic patients was unrelated to a defect in CCL18 binding to NK cells. Overall, our results suggest that some NK cell functions may be defective in allergic diseases

Activation of Eosinophils by NK cells.

<p>Autologous NK cells and eosinophils were cultured at the ratio NK cells: eosinophils (NK: Eos ratio) 0∶1 (i.e. eosinophils alone), 1∶1, 5∶1 and 10∶1 in the presence of interleukin-5 for 3 hours and 12 hours. (A) Gating strategy: live eosinophils were identified firstly on the basis of their size difference (FSC) and granularity (SSC) and secondly as Annexin V⁻ 7AAD⁻ cells. CD63 expression (black histogram) is shown on a representative experiment for the ratio NK cells: eosinophils (NK: Eos ratio) 0∶1 (i.e. eosinophils alone) and 10∶1. Isotypic control antibody is represented in dashed grey. Eosinophils were stained for CD63 (B), CD62L (C) and CD69 (D) expression. Results are shown for each donor and mean is indicated with the black line. One-way Anova (Friedman) tests were performed, followed by Dunn’s post tests. *p<0.05; ***p<0.001.</p

NK cell-induced eosinophil apoptosis involves mitochondrial reactive oxygen species (ROS).

<p>(A) After purification, NK cells and eosinophils were cultured in the presence of IL-5 for 12 hours at different NK:Eos ratios: 0∶1 (i.e. eosinophils alone); 1∶1; 5∶1; 10∶1. Eosinophils were identified on the basis of their size difference (FSC) and granularity (SSC). ROS staining was achieved using dihydroethidium (HE). Results are expressed as mean percentage of HE⁺ cells amongt the eosinophil population ± SEM. n = 12. Gaussian distributions were verified using D’Agostino and Pearson test, and One-way Anova test was performed, followed by Bonferroni’s post test. *p<0.05; **p<0.01. (B) Eosinophils were incubated with inhibitors of mitochondrial electron transport: rotenone and antimycin for 30 min at 37°C before 12 h co-culture with NK cells at the NK:Eos ratio of 10∶1. Results for each donor are shown and mean is indicated with the black line. Wilcoxon tests were performed. *p<0.05.</p

NK cells induce eosinophil apoptosis.

<p>After purification, NK cells and eosinophils were cultured in the presence of IL-5 for 3 and 12 h at different NK:Eos ratios: 0∶1 (i.e. eosinophils alone); 1∶1; 5∶1; 10∶1. Eosinophils were identified on the basis of their size difference (FSC) and granularity (SSC). Apoptosis and death of eosinophils was evaluated by Annexin/PI labeling. Dead eosinophils were defined as AnnexinV⁺ cells. Results are expressed as mean percentage of AnnexinV⁺ cells amongt the eosinophil population ± SEM. n = 16 (3 hours) or 18 (12 hours). Gaussian distributions were verified using D’Agostino and Pearson test, and One-way Anova tests were performed, followed by Bonferroni’s post tests. *p<0.05; **p<0.01; ***p<0.001.</p

MAPK and PI3K pathways are involved in eosinophil degranulation.

<p>AnnexinV⁻ eosinophils (A) and NK cells (B), cultured alone or together at the 10∶1 NK:Eos ratio, were stained with anti-ERK or anti-phosphorylated ERK (pERK) antibodies. Eosinophils and NK cells were identified on the basis of their size difference (FSC) and granularity (SSC). Live eosinophils were defined as AnnexinV⁻ cells. Gray full lines and black dotted lines represent control antibody staining for each cell type alone or in co-culture, respectively. Gray plain histograms and black full lines represent staining with antibody of interest for each cell type alone or in co-culture, respectively. One representative experiment out of three is shown. (C) Eosinophils were pre-incubated with DMSO as control or with inhibitors of signaling pathways for 5 min at 37°C: PD 98059 25 μM an inhibitor of ERK pathway, SB 203580 1 μM an inhibitor of P38 MAPK, SP 600125 20 μM an inhibitor of JNK pathway and LY 294002 10 μM an inhibitor of PI3K pathway for the NK:Eos ratio 10∶1. Results show percentage of CD63⁺ eosinophils for each donor and mean is indicated with the black line. One-way Anova (Friedman) tests were performed, followed by Dunn’s post tests. *p<0.05, **p<0.01.</p

NOD2 Signaling Circuitry during Allergen Sensitization Does Not Worsen Experimental Neutrophilic Asthma but Promotes a Th2/Th17 Profile in Asthma Patients but Not Healthy Subjects

Nucleotide-binding oligomerization domain 2 (NOD2) recognizes pathogens associated with the development of asthma. Moreover, NOD2 adjuvants are used in vaccine design to boost immune responses. Muramyl di-peptide (MDP) is a NOD2 ligand, which is able to promote Th2/Th17 responses. Furthermore, polymorphisms of the NOD2 receptor are associated with allergy and asthma development. This study aimed to evaluate if MDP given as an adjuvant during allergen sensitization may worsen the development of Th2/Th17 responses. We used a mouse model of Th2/Th17-type allergic neutrophil airway inflammation (AAI) to dog allergen, with in vitro polarization of human naive T cells by dendritic cells (DC) from healthy and dog-allergic asthma subjects. In the mouse model, intranasal co-administration of MDP did not modify the AAI parameters, including Th2/Th17-type lung inflammation. In humans, MDP co-stimulation of allergen-primed DC did not change the polarization profile of T cells in healthy subjects but elicited a Th2/Th17 profile in asthma subjects, as compared with MDP alone. These results support the idea that NOD2 may not be involved in the infection-related development of asthma and that, while care has to be taken in asthma patients, NOD2 adjuvants might be used in non-sensitized individuals