Project description:Neutrophilic granulocytes change their functionality and phenotype in response to cytokines. This adaptation to the micro-milieu, also called priming, is eminent for an orchestrated defense against infection. However, in chronic inflammation, this process may be accompanied by a steady maintenance of inflammation and tissue destruction. To explore this in more detail, neutrophilic granulocytes were obtained from the peripheral blood of healthy adult donors and primed with cytokines in vitro. The gene expression profile of these cells was compared to resting cells using nCounter technology.
Project description:Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.
Project description:The transcription factors that regulate gene expression in stimulated neutrophils are poorly characterized. Herein, we found that the genomic distribution of the myeloid lineage determining transcription factors PU.1 and C/EBPβ, as well as that of the histone modification H3K27Ac, which marks active chromatin, significantly change in human neutrophils treated with R848, a ligand of TLR8.
Project description:Compared to circulating neutrophils (NC cells), splenic neutrophils (NBH cells) have an activated phenotype and enhanced B cell-helper activity. The transcriptome analysis of splenic and circulating neutrophils was performed to verify whether the enhanced B cell-helper activity of splenic neutrophils correlated with a specific gene signature. Unstimulated neutrophils were FACS sorted from the peripheral blood and spleen of six adult healthy subjects for RNA isolation and Agilent analysis.