Abstract
BackgroundExperimental exposure of swine neutrophils to bacterial lipopolysaccharide (LPS) represents a model to study the innate immune response during bacterial infection. Neutrophils can effectively limit the infection by secreting lipid mediators, antimicrobial molecules and a combination of reactive oxygen species (ROS) without new synthesis of proteins. However, it is known that neutrophils can modify the gene expression after LPS exposure. We performed microarray gene expression analysis in order to elucidate the less known transcriptional response of neutrophils during infection.MethodsBlood samples were collected from four healthy Iberian pigs and neutrophils were isolated and incubated during 6, 9 and 18 hrs in presence or absence of lipopolysaccharide (LPS) from Salmonella enterica serovar Typhimurium. RNA was isolated and hybridized to Affymetrix Porcine GeneChip®. Microarray data were normalized using Robust Microarray Analysis (RMA) and then, differential expression was obtained by an analysis of variance (ANOVA).ResultsANOVA data analysis showed that the number of differentially expressed genes (DEG) after LPS treatment vary with time. The highest transcriptional response occurred at 9 hr post LPS stimulation with 1494 DEG whereas at 6 and 18 hr showed 125 and 108 DEG, respectively. Three different gene expression tendencies were observed: genes in cluster 1 showed a tendency toward up-regulation; cluster 2 genes showing a tendency for down-regulation at 9 hr; and cluster 3 genes were up-regulated at 9 hr post LPS stimulation. Ingenuity Pathway Analysis revealed a delay of neutrophil apoptosis at 9 hr. Many genes controlling biological functions were altered with time including those controlling metabolism and cell organization, ubiquitination, adhesion, movement or inflammatory response.ConclusionsLPS stimulation alters the transcriptional pattern in neutrophils and the present results show that the robust transcriptional potential of neutrophils under infection conditions, indicating that active regulation of gene expression plays a major role in the neutrophil-mediated- innate immune response.
Highlights
Experimental exposure of swine neutrophils to bacterial lipopolysaccharide (LPS) represents a model to study the innate immune response during bacterial infection
LPS stimulation alters the transcriptional pattern in neutrophils and the present results show that the robust transcriptional potential of neutrophils under infection conditions, indicating that active regulation of gene expression plays a major role in the neutrophil-mediated- innate immune response
To test the neutrophil transcriptional potential, global gene expression analysis was performed and the results indicated that the LPS-treated neutrophils increase their transcriptional activity by altering genes involved in different cellular processes including transcriptional regulation, cell signalling, cytoskeletal reorganization, etc
Summary
Isolation and stimulation of neutrophils Blood samples were collected from 4 healthy Iberian pigs at the local abattoir. RNA purification and microarray hybridizations RNA was isolated with RNeasy columns (Qiagen, Valencia, CA) based on the manufacturers protocol. The labeled cRNA is cleaned up, fragmented, and hybridized to Affymetrix Porcine GeneChip according to the manufacturers procedures (Expression Analysis Technical Manual, Affymetrix Inc., Santa Clara, CA). Differential expression and clustering was performed with Spotfire DecisionSite v 9.0 (SP2) as follows: feature values were obtained by a one-way analysis of variance (ANOVA) with a p-value < 0.05 and hierarchically clustered using UPGMA method and Pearsons correlation as the similarity measure. Since the Affymetrix Porcine GeneChip® is not fully annotated in all the features, it was re-annotated with Blast2GO [7] with a minimum E-value of 1010 and a minimum similarity of 50%. DEG were grouped into known biological functions, networks and canonical pathways based on human and rodent studies
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