Expression profiling of lung neutrophils
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA588438 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10423300.fastq.gz | Fastqsanger.gz | |||
| SRR10423301.fastq.gz | Fastqsanger.gz | |||
| SRR10423302.fastq.gz | Fastqsanger.gz | |||
| SRR10423303.fastq.gz | Fastqsanger.gz | |||
| SRR10423304.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Atglflox/flox (B6N.129S-Pnpla2tm1Eek/J), S100A8-cre+/- (B6.Cg-Tg(S100A8-cre,-EGFP)1Ilw/J) mice were obtained from The Jackson Laboratory. Atglflox/flox mice were bred to S100A8-cre+/- mice to generate Atglflox/WTS100A8-cre+/- mice, which were backcrossed onto Atglflox/flox mice to generate Atglflox/floxS100A8-cre+/- mice (Atgl neutrophils-specific knock out, Atgl-cKO). Age-matched littermate Atglflox/flox mice were used as wild-type (WT) controls. To compare of the gene expression of the lung-infiltrating neutrophils isolated from Atgl-cKO mice and their WT littermates, AT3-g-csf cells were injected into the fourth mammary fat pads of female WT and Atgl-cKO mice (10-week-old, n = 4/group). The AT3-g-csf cell line is based on a murine breast cancer cell line (AT3) derived from MMTV-PyMT tumors in the C57BL/6 background, and further constructed to overexpress granulocyte-colony stimulating factor (G-CSF) for induction of the host inflammatory condition. At day 10 (pre-metastatic stage), the mice were euthanized and then Ly6G+ neutrophils were isolated from lung by using anti-Ly6G MicroBead Kit (Miltenyi Biotec) following manufacturer’s instructions. The isolated neutrophils were analyzed by flow cytometry and the cells with a > 95% purity were used for the next procedure. Total RNA was isolated from neutrophils using the miRNeasy Mini kit (Qiagen) and the transcriptional profiles of neutrophils were analyzed by RNA sequencing.
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