Analysis of the bovine neutrophil transcriptome during glucocorticoid treatment.

The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo aging for 4 h with or without added Dex. Additional neutrophils were cotreated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of National Bovine Functional Genomics Consortium (NBFGC) microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4-h (untreated) neutrophils. Subsequent two-step mixed-model analysis detected (P < or = 0.05) 1,109 differentially expressed genes, of which contrast analysis indicated those that were independently responsive to aging (1,064), Dex (502), RU486 + Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 did not cluster well based on known function, and 634 clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex-responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound-healing actions of glucocorticoids.

Generation of EST and cDNA microarray resources for the study of bovine immunobiology.

Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG) at Michigan State University has developed a normalized bovine total leukocyte (BOTL) cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL) and the sequence information plus BLAST results made available through a web-accessible database (http://gowhite.ans.msu.edu). Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls) per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs). In total, hybridization signals on over 90 amplicons showed upregulation (> 3x) in response to Con A stimulation, relative to unstimulated cells. A second experiment with PBMCs from a different group of animals was performed to test reproducibility of microarray results. There was a high correlation between the 2 experiments (r = 0.72, P < 0.001). Resources described in this publication offer a highly efficient and integrated system to study gene expression changes in bovine leukocytes.

An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cows.

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with > 700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with > or = 1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.