Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic profiling of human neutrophils after single and sequential stimulation with PAF and fMLP

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: JENIFFER MENDES DA SILVA FREIRE

PROVIDER: E-MTAB-16930 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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)

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Transcriptomic profiling of human neutrophils after single and sequential stimulation with PAF and fMLP

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