Transcript
X. Revelo*, A. Kenny, N. Barkley, and M. WaldronDivision of Animal Sciences
University of Missouri, Columbia
Impaired capacity of neutrophils to produce reactive oxygen species, release extracellular
traps and express genes encoding for cytokines may contribute to altered immune
function in periparturient dairy cows
Neutrophil killing mechanisms
ExtracellularIntracellular
Reactive oxygen species (ROS)
Neutrophil extracellular traps (NETs)
Kennedy 2009 Brinkmann 2004
Objective
Investigate changes in gene expression associated with neutrophil dysfunction during the periparturient period in dairy cows- Neutrophil function:- Reactive oxygen species (ROS) production
- Extracellular trap formation (NET)
- Chemotaxis towards interleukin-8
- Phagocytosis of E. coli
-Gene expression:-Tumor necrosis factor-α mRNA
-Interleukin-8 mRNA
-Cytochrome-b mRNA
-Bactericidal/permeability increasing protein (BPI) mRNA
-Transcriptome (mRNA) shotgun sequencing
Research Methods
• Collection of blood Neutrophil function (n = 35)Late pregnancy: -58 ± 1.7 dPre-partum: -7 ± 0 dPostpartum: 6 d
Gene expression (n = 7)Late pregnancy: -58 ± 1.7 dPostpartum: 7 d
• Isolation of neutrophilsDensity gradient centrifugation
Research Methods
• Reactive oxygen species (ROS)
• Extracellular trap formation (NETs)
• Chemotaxis towards IL-8
• Phagocytosis of E. coli
• Gene expressionRT-PCR:
- Tumor necrosis factor-α- Interleukin-8- Cytochrome b- BPI
Illumina® RNA Sequencing
Altered neutrophil function in periparturient cows
Effect of period, P < 0.001 (n = 35) Revelo et al., 2011 (in preparation)
ROS Production Chemotaxis
Altered neutrophil function in periparturient cows
Effect of period, P < 0.001 (n = 35) Revelo et al., 2011 (in preparation)
NETs releasePhagocytosis
Reduced cytokine and ROS gene expression
Revelo and Waldron, 2011 (JDS, accepted)
Postpartum Late pregnancy Postpartum
Postpartum Postpartum Late pregnancy
Postpartum Late pregnancy
Postpartum Late pregnancy
Transcriptome analysis of bovine neutrophils
RNA-seq quality control (average of 14 libraries)Total reads 27,210,968
Reads converted 25,252,266
Perfect match 18,516,011
Matched reads 22,315,658
Unmatched reads 2,936,423
Matched bases 1,955,924,633
Unmatched bases mutations 2,640,876
Average read length 48
Reference length 2,670,102,999
Covered bases 259,076,285
Percent coverage, % 9.70
Percent mapped, % 88.37
Genes up-regulated postpartum (> 1.5 fold, P < 0.01) 43
Genes down-regulated postpartum (< 0.5 fold, P < 0.01) 423
Revelo et al., 2011 (in preparation)
Functional clustering: ↓NADPH/oxido-reductase
DAVID Bioinformatics Resources 6.7NADPH activity
Oxido-reductaseactivity
ROS pathway
• Periparturient neutrophil function and mRNA expression • ↓ Reactive oxygen species
• ↓ Chemotaxis
• ↓ Phagocytosis
• ↓ Interleukin-8 mRNA
• ↓ Tumor necrosis factor-α mRNA
• ↓ Cytochrome-b, alpha polypeptide mRNA
• ↓ Bactericidal/permeability increasing protein mRNA
• Transcriptome analysis reveals clusters of genes with reduced expression in neutrophils during postpartum
Summary
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