Project description:Coding RNA expression of blood eosinophils from mepolizumab- or omalizumab-treated patients stimulated ex vivo with IL-33 for 6 hours and of directly processed non-stimulated blood eosinophils from the same samples (paired design)
Project description:Aim of the experiment was to assess differences in expression of coding RNA between bone marrow eosinophils arising in IL-5 knock-out mice (C57BL/6-Il5tm1Kopf/J) or wild-type littermates and their response to cytokine stimulation. Bone marrow eosinophils were sorted by FACS submitted to RNA-seq immediately or after 4 hours of stimulation with IL-5 and IL-33 in culture plates.
Project description:We conducted single-cell gene expression analysis of neutrophils from mouse tumors with and without microbial treatment to investigate neutrophil response in the tumor microenvironment (TME). Briefly, tumor-infiltrating Ly6G+CD11b+ neutrophils were isolated from unmanipulated tumors (resting), tumors treated with a vehicle control (control), and tumors treated with S. aureus bioparticles (stimulated) 24 hours after treatment. To survey the whole spectrum of the TME, we also isolated and sequenced non-neutrophil leukocytes in each sample.
Project description:RNA-seq comparison of coding RNA expression in blood eosinophils from patients with severe allergic eosinophilic asthma treated with mepolizumab with those of healthy controls and matched severe asthmatic patients receiving omalizumab.
Project description:To dissect the precise roles of ST-DC2 clusters and ST-iDC3 clusters in T-cell activation, we co-cultured each cluster separately with autologous memory CD4pos T-cells. FACS-sorted ST-DC2 clusters and ST-iDC3 clusters from synovial tissue of active RA or remission RA were co-cultured for 5 days with FACS-sorted autologous blood memory CD4pos T-cells in the presence of anti-CD3 stimulation to mimic TCR activation. We investigated the frequency and phenotype of the resultant co-culture T-cell populations by carrying out single cell sequencing and analysing the output. It was observed that the DC2 and iDC3 group could differentially support distinct T-Cell populations in both frequency, and transcriptional profile.
Project description:SPP1 stimulation of human leukocytes drives proinflammatory monocyte activation and differentiation of dysregulated CD274(PDL-1)pos neutrophil phenotype.
Project description:Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma, and thus are considered an important factor in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations, in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) followed by protein identification by MS, and by label-free LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL 3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69 expression. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater than additive effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.
Project description:4 groups of mice : Control, antibiotics, antibiotics + 4days of recolonization, antibiotics + 4days of recolonization + Enterocloster clostridioformis. Tumor draining lymph node were harvested after CFSE injection were harvested and CFSE+ cells were sorted and proccessed in order to generate single cell RNA-sequencing using BD Rhapsody mouse immune response targeted panel. Groups were barcoded using BD Rhapsody Multiplexing Kit.
Project description:We tested the role of distinct STM populations in the modulation of synovial tissue environment in ex vivo STM-FLS micro co-cultures. We first FACS-sorted total synovial tissue fibroblasts as we described previously from biopsies of RA patients. FLS were seeded at 3000/well alone or in contact with either 3000/well FACS-sorted MerTK/CD206neg or MerTK/CD206pos STMs from patients with active or remission RA respectively for 48h. The modulatory effect on the FLS was evaluated by comparing their expression of 446 immune/stromal genes (scRNAseq BD Rhapsody) 32,141 FLS were evaluated. FLS cells cultured alone exhibited 4 distinct activation states: FLS cluster 1 (FLS1) expressed extracellular matrix proteins (e.g. COL1A1, COL1A2) and TGFb (TGFBI, TGFB3); FLS2 expressed cell adhesion molecules (e.g. ITGB2, SELPLG); FLS3 expressed receptors for TGFb and resolvin (e.g.CMKLR1 and TGFBR1); and FLS4 expressed high levels of glycolytic enzymes and proliferation markers (e.g. LDHA, PGK1, ENO1 and PCNA). Interestingly, upon co-culture with MerTK/CD206neg an additional fifth cluster (FLS5) emerged with inflammatory properties In contrast to inflammatory effect of the MerTK/CD206neg STMs on FLS, the MerTK/CD206pos population, especially that isolated from biopsies of RA patients in sustained disease remission, induced repair response of FLS as manifested by increased expression of collagens (e.g. COL1A) and TGFb response genes (e.g. TGFBI) Importantly, MerTK/CD206neg and MerTK/CD206pos STMs isolated from the biopsies of the same patient (either with active RA or RA in remission) elicited these divergent FLS responses. These suggest that these two distinct STM populations play opposing role in synovium (pro-inflammatory versus protective).
Project description:As opposed to their well-characterized contributions to inflammatory processes, tissue eosinophils are now also thought to contribute to immune homeostasis at mucosal sites such as the gut. Yet, whether such steady-state eosinophils exist in the lung is currently unclear. In this project, we identified and characterized lung resident eosinophil and also studied what happen to these cell during a mouse model of allergic inflammation (House dust mite (HDM) exact administrations). We compared the transcriptional profile of lung resident eosinophil from naive mice (rEOS ss) or from HDM-treated mice (rEOS i) and of inflammatory eosinophil (iEOS) from HDM-treated mice.