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

Neutrophils are key immune cells

Killing

Chemotaxis

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

Pathway analysis: ↓ROS Gene Expression

KEGG pathway database

Down-regulated, P < 0.01

• 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

Take home message

Questions