Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable. A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen). 24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS, 25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent), and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA. There are no batches within each experiment - the samples in each group are exchangeable. Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays. Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.

Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in unstimulated and fMLP-stimulated neutrophils derived from mouse bone marrow, we performed micorarray analysis of gene expression in unstimulated wild type (N=3), unstimulated lcn2 knockout (N=3), fMLP-stimulated wild type (N=2) and fMLP-stimulated lcn2 knockout (N=2) neutrophils. Upon stimulation, neutrophils were treated by fMLP at 10 micromolar for 20 minutes at 37 centigrade.

Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in regulating miRNA expression profiling in neutrophils derived from mouse bone marrow, we performed microarray analysis of miRNAs in wild type (N=2) and lcn2 knockout (N=2) neutrophils.

Dataset Information

Homo sapiens

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets