Project description:Analysis of neutrophil proteomic alterations induced by migration towards inflamed joints in juvenile idiopathic arthritis (JIA). In this experiment neutrophil proteomes were investigated after migration towards JIA synovial fluid in an in vitro model of a synovial membrane, compared to neutrophils incubated in synovial fluid without migration. The migration model consisted of transwell inserts with human knee synoviocytes on the undersides and HMEC endothelial cells on the insides, placed in wells containing medium with 10 % JIA synovial fluid.
Project description:Neutrophils, crucial player in inflammation, present a significant challenge in comprehensive molecular characterization due to limited availability (~1,000 cells) in inflamed sites like steady state infiltration. Prior proteomic studies typically required millions of neutrophils, posing difficulties when dealing with limited samples. This study introduces a ‘low-cell proteome’ workflow for neutrophils, integrating S-trap micro column-based digestion with DIA PASEF to investigate their functional dynamics within individual inflamed site.This method enable protein profiling of 1,000 isoated resting circulatory neutrophils with broad dynamic range in a reproducible manner.Furthermore, we applied this integrated approach to investigate the change in neutrophil proteome under different physiological and pathological conditions, including brain infiltration following stroke in mice and transmigration to the oral cavity post-treatment with tabasco in humans. Our comprehensive proteomic analysis provides insights into the distinct behavioral responses of neutrophils in these inflammatory contexts compared to their circulating counterparts.
Project description:We performed a systematic investigation of seven pharmacologic inhibitors (calpeptin, D-pantethine, Y27632, cytochalasin D, GW4869, R5421, and Nexinhib20) on quantitative and qualitative EV properties derived from resting neutrophils. We measured EV numbers, size distribution, total lipid, protein, and RNA content. We also analyzed the EV morphology by cryo-EM and EV cargo qualitatively by mass spectrometry and RNA sequencing (mRNA, miRNA).
Project description:We performed a systematic investigation of seven pharmacologic inhibitors (calpeptin, D-pantethine, Y27632, cytochalasin D, GW4869, R5421, and Nexinhib20) on quantitative and qualitative EV properties derived from resting neutrophils. We measured EV numbers, size distribution, total lipid, protein, and RNA content. We also analyzed the EV morphology by cryo-EM and EV cargo qualitatively by mass spectrometry and RNA sequencing (mRNA, miRNA).
Project description:This study evaluated the miRNA expression profile of human neutrophils under resting conditions and after stimulation with PAF and fMLP, applied alone or sequentially. The objective was to determine whether distinct inflammatory stimuli and their order of exposure induce different miRNA expression patterns in neutrophils. Neutrophils from healthy donors were assigned to five conditions: control, PAF, fMLP, PAF followed by fMLP (PF), and fMLP followed by PAF (FP). Total RNA was extracted and hybridized to the Affymetrix miRNA 4.0 Array platform for expression analysis.
Project description:biopsies from the most inflamed area were sequenced where applicable. FACS-sorted cell populations: N=neutrophils; M=mononuclear phagocytes; S=stromal cells; E=eosinophils