MiRNA expression profiling of human neutrophils after single and sequential stimulation with PAF and fMLP
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: JENIFFER MENDES DA SILVA FREIRE
PROVIDER: E-MTAB-16957 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Human neutrophils isolated from peripheral blood of five healthy male donors were analyzed by one-color microarray to investigate transcriptional changes induced by single and sequential stimulation with platelet-activating factor (PAF) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). The experiment included five conditions: unstimulated control, PAF, fMLP, PAF followed by fMLP, and fMLP followed by PAF, totaling 25 samples. This design was used to evaluate whether the order of exposure to endogenous and exogenous inflammatory stimuli influences neutrophil transcriptional responses. Total RNA was extracted after stimulation and hybridized to the Clariom S Human Array according to the manufacturer’s protocol. The study aimed to characterize stimulus-dependent and order-dependent transcriptional profiles in human neutrophils.
2026-04-19 | E-MTAB-16930 | biostudies-arrayexpress
Project description:Neutrophil accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. Based on recent evidence that mucosal metabolic changes influence disease outcomes, we hypothesized that transmigrating neutrophils influence the transcriptional profile of intestinal epithelia. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by neutrophil-epithelial crosstalk. Real-time O2 sensing indicated that transmigrating neutrophils rapidly deplete microenvironmental O2 sufficient enough to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the relative contribution of neutrophils and the respiratory burst to M-bM-^@M-^\inflammatory hypoxiaM-bM-^@M-^] in vivo. Gp91phox-null mice, which mirror human chronic granulomatous disease, developed accentuated colitis compared to control with exaggerated neutrophil infiltration and diminished inflammatory hypoxia. In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils modulates the host response to inflammation. Likewise, the respiratory burst contributes fundamentally to localized O2 depletion, resultant microenvironmental hypoxia and effective inflammatory resolution. Two models were employed, direct and indirect. The M-bM-^@M-^\DirectM-bM-^@M-^] migration model entailed establishing a chemotactic gradient (using fMLP) across monolayers of T84 intestinal epithelial cells grown on the underside of permeable supports (3um pore). Neutrophils (PMN) were induced to migrate in the physiologically relevant the physiologically relevant basolateral-to-apical direction. Following migration, T84s were rested in complete media and 2hrs later harvested for RNA isolation. In the Indirect model, PMN were applied to T84s as with the direct model. After migration, conditioned supernatants were collected, cells pelleted and supernatants filtered through 0.2um pore. Conditioned supernatants were transferred to naive T84 monolayers for 2hrs, followed by RNA harvest. Each model was exposed to neutrophils (PMN) or not. All monolayers contained chemotactic peptide fMLP on the apical side. Total of 12 samples, 4 conditions in triplicate: Direct migration without neutrophils (T84 +fMLP -PMN), Direct migration with neutrophils (T84 +fMLP +PMN), Indirect migration without neutrophils (T84 +fMLP -PMN), Indirect migration with neutrophils (T84 +fMLP +PMN)
2012-10-31 | E-GEOD-39681 | biostudies-arrayexpress
Project description:Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. Herein, we used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiologic basolateral-to-apical direction to a gradient of fMLP. Control wells were treated with fMLP alone. Indicated wells were pretreated with an anti-CD47 blocking antibody, which exacerbates epithelial injury. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifest by restoration of transepithelial resistance (TER) and re-epithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. This pathway represents a potential therapeutic target to accelerate physiologic recovery in inflammatory lung diseases. Total of 20 samples: Calu-3 cells that were untreated, treated with fMLP, treated with fMLP and neutrophil transmigration, and treated with anti-CD47 antibodies, fMLP, and neutrophil transmigration; cDNA was pooled from n=3 experiments; No biological replicates
2011-08-27 | E-GEOD-31697 | biostudies-arrayexpress
Project description:New alkyl-phospholipids (APLs) that are structurally derived from the platelet-activating factor are promising candidates for anticancer treatment. After the incorporation into cell membranes, APLs are able to interfere with a wide variety of key enzymes implicated in cell growth, motility, invasion and apoptosis. Besides the prototype edelfosine, we presented a novel group of APLs, glycosidated phospholipids that efficiently inhibit cell proliferation. Two members of this group, Ino-C2-PAF and Glc-PAF, display high efficacy and low cytotoxicity in immortalized non-tumorigenic skin keratinocyte cell line HaCaT. However, the influence of APLs on the transcription of the whole genome is still unknown. Here, using Agilent cDNA microarray technology, we compared global gene expression profiles of HaCaT cells treated with edelfosine, Ino-C2-PAF or Glc-PAF with the profile of control cells. The influence of APLs on the transcriptional profile of immortalized keratinocytes (HaCaT) was analyzed treating HaCaT cells with respectively 5 M-BM-5M Ino-C2-PAF, Glc-PAF and edelfosine for 24 h. Control cells were left untreated. Three independent experiments were performed for each condition.
2011-03-01 | E-GEOD-20722 | biostudies-arrayexpress
Project description:In this study, we explored transcriptional complexity in human neutrophils, cells generally regarded as nonspecific in their functions and responses. We studied distinct human disease phenotypes and found that, at the gene, gene isoform, and miRNA level, neutrophils exhibit considerable specificity in their transcriptomes. These findings were particularly striking for isoform usage. Thus, even cells whose responses are considered non-specific show tailoring of their transcriptional repertoire toward specific physiologic or pathologic contexts. These findings have important implications for our understanding of the link between gene expression and disease phenotypes. Total RNA was extracted from isolated neutrophils from patients with JIA, from patients with cystic fibrosis and from healthy controls.
2015-09-18 | E-GEOD-71010 | biostudies-arrayexpress
Project description:Functional role of platelet-activating factor (PAF) induced arachidonic acid release
2009-10-31 | E-TABM-472 | biostudies-arrayexpress
Project description:Neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow. We used microarray to globally analyze gene expression in aged neutrophils and characterize the inflammatory programs that are activated during the aging process in the circulation. Control, aged and activated neutrophils were sorted directly from mouse blood for RNA extraction and hybridization on Affymetrix microarrays. We transfused whole blood and harvested donor neutrophils marked by the CD45.1 allele 6h later to derive neutrophils that had truly aged in vivo. Sorted neutrophils were compared to control donor neutrophils that had been transferred for only 10 min. Additionally, we harvested circulating neutrophils from TNF-? treated mice for comparison with neutrophils activated by systemic inflammation.
2015-09-24 | E-GEOD-69886 | biostudies-arrayexpress
Project description:Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3 and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4 and 14‐3‐3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.
2024-01-26 | PXD046791 | Pride
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.
2013-01-31 | E-GEOD-43889 | biostudies-arrayexpress
Project description:New alkyl-phospholipids (APLs) that are structurally derived from the platelet-activating factor are promising candidates for anticancer treatment. After the incorporation into cell membranes, APLs are able to interfere with a wide variety of key enzymes implicated in cell growth, motility, invasion and apoptosis. Recently, using Agilent cDNA microarray technology, we could demonstrate that the glycosidated APL Ino-C2-PAF inhibited the expression of several genes associated with immune response and inflammation in immortalized keratinocytes HaCaT. Here, we analyzed the impact of Ino-C2-PAF on the gene expression profile of HaCaT cells treated with several pro-inflammatory cytokines (IL-1M-NM-1, IL-17, IL-22, TNF-M-NM-1 and oncostatin-M). The influence of Ino-C2-PAF on the transcriptional profile of immortalized keratinocytes (HaCaT) was analyzed treating HaCaT cells with 5 M-BM-5M Ino-C2-PAF in the presence of a mixture of pro-inflammatory cytokines (IL-1M-NM-1, IL-17, IL-22, TNF-M-NM-1 and oncostatin-M) for 24 h. Control cells were left untreated. Three independent experiments were performed for each condition, except for control cells where only two experiments were performed. Control cells were mainly necessary to demonstrate the efficiency of this in vitro inflammation model for keratinocytes.
2013-01-01 | E-GEOD-37361 | biostudies-arrayexpress