Disease-Associated SNPs From non-Coding Regions in Juvenile Idiopathic Arthritis Are Located Within or Adjacent to Functional Genomic Elements of Human Neutrophils and CD4+ T Cells [RNA-Seq]
Ontology highlight
ABSTRACT: We sequenced mRNA from 3 neutrophil cells taken from 3 male adult to generate the gene expression profile of human neutrophil cells Examination of mRNA levels in human neutrophils.
Project description:We applied ChIP-Seq on two histone marks: H3K4me1 and H3K27ac in healthy human neutrophils. After peak calling, we obtained the peak regions enriched with H3K4me1 and H3K27ac histone marks and identifed aciive enhancers (H3K27ac+/H3K4me1+) and H3K27ac active enhancers (H3K27ac+/H3K4me1-) in human neutrophils and checked whether those enhancers are located in the LD blocks of 22 SNPs associtated with Juvenile Idiopathic Arthritis. Identification of active enhancers in human neutrohils
Project description:IFNG decreases incidence of infections in chronic granulomatous disease (CGD) without affecting inability of CGD neutrophils to generate essential microbicidal oxidants. Neutrophil (PMN) function, gene expression, and biochemical parameters were measured off IFN-γ and 10-12 hours after 1st and 4th doses of IFN-γ administered to nine CGD patients. Non-directed motility and bactericidal activity were increased after treatment with IFN-γ; ingestion and O2- generation remained unchanged. Treatment decreased expression of 483 genes and increased 386. Genes (11) associated with PMN activity were upregulated. Genes not routinely associated with neutrophil function were increased including MHCI and II, guanylate binding proteins, and an enzyme synthesizing a NOS cofactor. CD11b expression, f-Actin assembly, and CD 274 antigen were increased after treatment as was NO in PMN lysates. NETs formation after ingestion was increased off IFN- but moved toward normal after administration. IFN- provides neutrophils with strategies to compensate for the deficiency found in CGD.
Project description:Whole blood and purified leucocyte populations were compared to determine which cell types the whole blood interferon-inducible signature, found in both tuberculosis and sarcoidosis, was present in. This study agreed with previous publication that the neutrophil was the dominant cell in tuberculosis and also showed it was the dominant cell type in sarcoidosis. There were 5 patients in each group of healthy controls, tuberculosis and sarcoidosis. The cell types were whole blood, PBMCs, monocytes (CD14), T cells (CD8 and CD4) and neutrophils (CD15).
Project description:Gene expression profiles were obtained from 17 children with rheumatoid factor negative (RF-) polyarticular juvenile idiopathic arthritis (JIA). The disease was inactive in all patients at visit 1. Thee disease remained inactive at visit 2 for 9 patients, while the other 8 patients were experiencing a disease flare at visit 2. The goal of the study was to better understand the underlying molecular biology of flares in JIA Total RNA was obtained from PBMC at two different times in the course of disease 2 samples from each of 17 patients were collected and analyzed different times in the course of the disease
Project description:During systemic inflammation, different neutrophil subsets are mobilized to the blood circulation. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright) based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but the underlying mechanism is largely unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes that are relevant for their functions. Neutrophil subsets were isolated by FACS sorting from the blood of healthy volunteers who were administered a single dose of lipopolysaccharide (LPS). The transcriptome was determined by microarray. The mobilized neutrophil subsets were characterized by specific transcriptome profiles reflecting their phase in neutrophil lifespan. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. This was confirmed by stimulation of peripheral neutrophils with IFNgamma. These cells acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that the suppressive phenotype of the neutrophil subset is induced by IFNgamma. Specific stimulation of neutrophils might have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease. After LPS infusion, blood was taken at t=0 and t=4 hours. Neutrophils were FACS sorted based on CD16 and CD62L expression. Gene expression of neutrophil subsets was assessed relative to t=0 as control.
Project description:Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow can be shaped within the circulation remains vague. Phenotypic changes of circulating neutrophils caused by systemic inflammation are thought to result from several factors, including a “left shift” of the neutrophil compartment towards younger bone marrow-derived subsets. However, experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile the human and murine neutrophil transcriptome and proteome during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to plastic shifts dominated by conserved upregulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB-signaling dependent upregulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Importantly, blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by upregulation of distinct effector molecules upon infection. In summary, these data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation. This adds to the understanding of competing modes of adaptation to inflammatory challenges by the neutrophil compartment.
Project description:Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson’s disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Monitoring pRab10 can thus serve as a readout for LRRK2 activity; however, no sufficiently accurate assay to quantify pRab10 levels for drug target engagement or patient stratification exists. Here, we developed an ultra-sensitive targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in human neutrophils before and after LRRK2 inhibition. Compared to healthy controls, neutrophils of LRRK2 G2019S and VPS35 D620N carriers robustly display 1.4-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.
Project description:We aimed to investigate the effect of CD40L on neutrophil development by comparing the transcription profile of wild-type and knockout mice. Thus, we performed RNA-sequencing of bone marrow neutrophils from wild-type mice (C57BL/6) and CD40LG knockout mice.
Project description:Lung neutrophils are causally associated with IAV-induced disease severity. Less is known about the repertoire of lethal IAV-associated neutrophil proteins or about how global changes in different neutrophil compartments are coordinated following lethal IAV infection. Here, we use semi-quantitative proteomics to characterize dynamic alterations in BM, blood and lung neutrophils at homeostasis or following a lethal IAV infection, with a secondary aim of identifying lung neutrophil-derived proteins which are selectively induced following IAV infection. Our findings identify bone marrow neutrophil maturation as the key site of anti-viral activity induction, with further upregulation or release of both anti-viral and antimicrobial effectors occurring following lung tissue infiltration.