Weaning is a multifactorial stressor, often encompassing separation from the dam and peers, mixing with unfamiliar animals, housing, novel handling and a change in diet and environment. The results of the present study demonstrate that the leukocyte transcriptomic environment is profoundly altered by weaning stress, at least up to 7 days post-weaning and that a number of processes are transcriptionally activated to increase immune cell adhesion and migration, potentially serving to increase immune surveillance following exposure to weaning stress.
Neutrophilia resulting from a stressor, as seen in this study, has been frequently reported in both cattle [5, 6, 9, 10, 19] and humans . This can partly be attributed to a surge of cortisol [35, 36] which may result in a series of physiological alterations to neutrophil function including the release of a large number of immature neutrophils from the bone marrow [37, 38], an increase in neutrophil chemotaxis  and a reduction in the neutrophil apoptotic rate [35, 36, 40, 41]. However, circulating cortisol concentrations peak early in the stress response and it is probable that the infrequent sampling intervals employed in the current study likely missed the true peak [42, 43].
It has previously been reported that stress-induced cortisol surges inhibit CD62L (L-selectin) expression on bovine neutrophils, thus reducing endothelial adhesion and subsequent migration to sites of infection [19, 44, 45]. However, several other bovine transport , castration  and weaning  studies have failed to identify this suppression transcriptionally. Furthermore, L-selectin expression may not be necessary to maintain leukocyte rolling as platelets have been reported to re-establish leukocyte trafficking in CD62L deficient mice . It is clear that a number of genes involved in integrin binding are up-regulated in weaned calves and that, while no difference in the expression of CD62L was identified, several other important integrins involved in cell adhesion and migration were up-regulated, in addition to a clear activation of platelets. The induction of a number of genes involved in transmembrane transport, including the glucose carrier family SLC5, the potassium dependent sodium/calcium exchanger SLC24, and the sodium and chloride dependent neurotransmitter transporter, SLC6A15, demonstrates an increased role for transmembrane transport of small molecules to leukocytes , and further work is necessary to examine if this may be involved in their functional activation.
Cytokines play an important role in the immune response, primarily functioning as inflammatory mediators of the innate immune system . The transcriptional induction of genes involved in chemotaxis provides a clear mechanism by which leukocyte function was altered. Chemokines are primarily involved in the recruitment of immune cells to inflammatory sites [50, 51], and their increased expression indicates that weaning stress results in transcriptional alterations that may enhance leukocyte recruitment and activation. Despite a preponderance of differentially expressed cytokine transcripts known to interact directly with neutrophils [52–54], it is likely many of these cytokines also influenced a number of other cell populations including lymphocytes, monocytes and platelets [55–58]. This is perhaps best demonstrated by the large induction of the GPCR signalling pathway with a number of regulators of G-protein signalling (RGS) genes remaining up-regulated throughout the course of this study, suggesting the rate of GPCR signalling was increased . Transcriptional signatures suggest neutrophils, monocytes and lymphocytes may have had an enhanced capability to exit the vasculature and increase immune surveillance through an up-regulation of a number of genes responsible for chemotaxis and integrin binding, indicating enhanced leukocyte function in response to stress, However, it would be of interest in future studies to validate this hypothesis via cell adhesion and migration assays in order to confirm the corresponding physiological state.
Inflammation is tightly linked to haemostasis, an important innate immune defence mechanism primarily intended to arrest the bleeding process , although it has recently been shown that platelets have additional functionality and interact with cells of the innate immune system via secretion of cytokines, chemokines and other inflammatory mediators [61–64]. In the current study, pathway analysis suggests that the process of haemostasis was activated as a result of weaning, and based on this activation, may have resulted in increased platelet aggregation, reducing the number of platelets freely available in circulation, as identified in weaned calves on day 1. A number of genes involved in collagen synthesis were differentially expressed with the overwhelming majority down-regulated in weaned animals, both in relation to control calves and the pre-weaning baseline. An early reaction to tissue damage is the release of collagen into blood vessels where it acts as a potent agonist of platelets and other leukocytes . This is mediated by cytokine induced collagenase secretion along with the down-regulation of collagen mRNA by cortisol [66–68], aiding in the rapid migration of leukocytes through the extracellular matrix (ECM) to sites of inflammation . An examination of genes involved in the extracellular region revealed the presence of a number of genes responsible for the activation and coagulation of platelets [70–72], in addition to platelet derived chemokines, such as CCL2 , CXCL5 , CXCL7  and platelet-derived growth factor C (PDGFC)  which may have facilitated the recruitment of neutrophils, monocytes, lymphocytes and other leukocytes to susceptible tissues, signifying that stress can transcriptionally activate an acute inflammatory response through interactions with platelets and the haemostatic system.
A large number of genes were differentially expressed on d 7 in weaned animals when compared with baseline expression values. While we have previously reported a detectable signature of inflammatory candidate genes in bovine leukocytes 7 days following weaning , it was unexpected that the expression of such a large number of genes (>300) would remain altered for so long following weaning, although it has been previously reported that a number of physiological parameters, including plasma fibrinogen and haptoglobin concentrations, and total leukocyte number had not returned to baseline in weaned calves 35 days following weaning . Results from the current study indicate that weaning can have a profound impact on the leukocyte transcriptome for at least 7 days post-weaning and suggests that the stress of weaning can have far reaching consequences for the homeostasis of the transcriptome. The majority of these genes were up-regulated and principally involved in cell signalling and function with a number of cytokines involved in the Th1 response . An increase in the magnitude of the stressor can quickly result in deleterious effects  as a prolonged inflammatory response can cause severe tissue damage [41, 77].
The greatest induction of genes in the control calves also occurred on d 7, a rather unexpected result given that these animals were not additionally exposed to any other stressor. However, the profile for these calves was much different than that of the weaned animals with the cytokine signalling, transmembrane transport, haemostasis and GPCR signalling pathways all having returned to baseline by d 7. Rather, almost 86% of the genes, which related to DNA replication and the cell cycle, were down-regulated on d 7 in relation to the pre-housing baseline. While it is clear that housing results in a molecular stress response on d 1 and 2, the results of d 7 require further examination as a similar effect was not found in the weaned treatment, despite these calves also being housed in addition to weaning. However, further research is required to assess the long term effect of housing stress in weaned calves. In the first 2 days following housing, a similar stress induced gene expression profile is present in both control and weaned calves, with a greater magnitude of induction in weaned calves exposed to the accumulative stress of housing, weaning and social reorganisation.