Uterine disease and disrupted fertility is the single biggest factor limiting the international dairy industry, with costs estimated at €1.4 billion within the European Union alone
. Understanding the role of the immune response in driving bacterial clearance postpartum, but also in restoring homeostasis, is likely to lead to new methods for enhancing fertility.
At both a cellular and a molecular level, there is increasing evidence for an intimate role of the immune response during involution. Neutrophil influx into the endometrium is a common mechanism associated with beneficial bacterial clearance, although evidence suggests that their phagocytic activity may be reduced in disease susceptible animals
. Recent research has shown the activation of many immune activation pathways (TLRs)
[11–13], inflammatory genes
, antimicrobial peptides and acute phase proteins
[6, 10] during the early postpartum period. Expression of the receptor for bacterial lipopolysaccharide, TLR4 for example, has been shown to be temporally elevated in the postpartum uterus during involution
[6, 13], between high and low fertility cows
 and in endometrial epithelial and stromal cells in response to LPS in vitro.
However, unregulated inflammation can lead to disease
, therefore a balanced inflammatory immune response is key to sufficient bacterial clearance and the restoration of an endometrial environment capable of supporting a new pregnancy
. This resolution of inflammation and the pathways involved in restoration of uterine homeostasis have not been extensively explored in healthy cows. Significantly elevated endometrial inflammatory gene expression (TNF, IL1, IL6 and IL8) has been previously documented in cows with endometritis at various time points postpartum
, supporting the hypothesis that dysregulation of the immune response (possibly as a consequence of inadequate bacterial clearance) is a contributory factor to disease development.
Endometrial inflammation was graded based on the number of leukocytes present in the biopsy and candidate gene expression analysis showed significant temporal differences in this limited gene dataset (such as IL1β, TNF, IFNG, IL8, TAP, DEFB5) between the two time points across involution, as previously described
. The different ontological classes of differentially expressed genes (cytokines, antimicrobial peptides, acute phase proteins) led us to hypothesise that temporal genome wide differential gene expression profiles would shed light on the pathways involved in both the activation of an inflammatory immune response early postpartum but importantly, also on the pathways involved in the regulation of inflammation and the restoration of homeostasis in these healthy cows. In the current study using the same animals, we use next generation sequencing to define the genome wide temporal changes in gene expression at two time points during involution in the postpartum cow.
Approximately only 27% of bovine genes are currently represented in the KEGG database, and therefore using a P-value cut-off of < 0.05, 2,856 genes were found to be significantly differentially expressed between endometrial biopsies from the same animals across time points, 15 DPP and 30 DPP. Using increased stringency of an adjusted P < 0.1, over 1,100 genes were found to be significantly differentially expressed. In both sets of results, the immune system was predominantly represented by enriched pathways at 15 DPP. These pathways included T cell receptor signalling, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, graft-versus-host disease and allograft rejection. This result concurs with related studies which have examined the expression of a limited number of candidate immune genes to show the activation of an inflammatory immune response in the postpartum endometrium
[5, 6, 10, 11].
However, the majority of the differentially expressed genes detected in this study were significantly increased at the 30 DPP time point. The resolution of inflammation, as defined by histopathological assessment in these cows
, was supported by the significant decrease in expression of immune pathways as involution proceeds. At this time point (30 DPP) enriched pathways, such as focal adhesion, wnt signalling and ECM-receptor interaction, were associated with tissue regeneration and proliferative activity, reflecting repair processes. This complete temporal change in the endometrial transcriptomic profile from a pro-inflammatory immune response phenotype to a tissue regenerative profile shows rapid but transient immune induction in the uterus to clear bacteria and reduce associated inflammation within a 2 week period in healthy cows. It is likely that changes in both resident and migratory cell populations between time points in response to bacterial infection and during involution account for some of the endometrial transcriptome differences detected. However, expression of a number of leukocyte associated markers (CD11d, CD14, CD16, CD18, CD45, CD48 and CD53) were not significantly different using our stringent selection criteria. Interestingly, CD62L - a cell surface adhesion molecule found on the surface of neutrophils was significantly elevated in expression 15 DPP (Log2 4.3 fold, P < 0.01), which correlates with the increased neutrophils detected in these animals using histology. However CD62L was not significantly differentially expressed using an adjusted P-value of < 0.1.
Endometrial receptivity has previously been associated with an increase in extracellular matrix (ECM) remodelling pathways
[26, 27], which is also one of the top enriched pathways 30 DPP in this study. A cancer associated pathway is another of the top enriched pathways 30 DPP which highlights the proliferative capacity of the endometrium in the current study. Gene-expression patterns linked with the regeneration of damaged tissue closely resemble that of highly malignant tumors, as there is significant enrichment of genes involved in cell proliferation in both instances
. In addition to cell proliferative processes, the endometrium is also undergoing regulation of cell growth and differentiation, and tissue homeostasis and repair 30 DPP, evident by the enrichment of the wnt signalling pathway
Five candidate genes involved in this temporal transcriptomic change (SAA1/2, IGF1,
GATA2, SERPINA14 and SHC2), were found to be significantly differentially expressed 30 DPP. The elevation of SAA1/2 expression 30 DPP, suggests it has a role in postpartum inflammatory resolution in the endometrium of healthy cows. Serum amyloid A (SAA) is an acute phase protein (APP) produced and released by hepatocytes but it is also expressed in extrahepatic bovine tissues
[31, 32], constitutively in healthy endometria
[33, 34] and during inflammation
[6, 10]. Studies have shown that SAA functions to preserve tissue maintenance and homeostasis
[35, 36] and SAA1/2 in particular has been shown in murine studies to be involved in the provision of immune homeostasis in mucosal tissue
. The elevated expression of SAA1/2 30 DPP may be indicative of the re-establishment of immune homeostasis later in involution as inflammation resolves.
Results also show the significant increased expression of GATA2 as involution progresses in the postpartum uterus. Gene expression of interferon-tau (IFN-τ), a luteotrophic molecule, is regulated by the expression of GATA2 in the bovine trophoblast
, and is also expressed in endometrial epithelial cells during the peri-attachment period of the conceptus in sheep
. GATA2 has also been recently shown to regulate the gene expression of endomucin, which is critical for cell growth, migration and angiogenesis, to ensure endothelial cell maintenance and physiological function
At systemic and local levels the insulin growth factor (IGF) system is implicated in endometrial repair and healing during involution
[41, 42]. Decreased expression of IGF1 has been observed in the previously gravid compared to the non-gravid uterine horn of the same cow 14 DPP
. In the present study we compared the progression of involution in the previously gravid uterine horn to a later stage of involution in the same horn and demonstrated that the expression of IGF1 was elevated 30 DPP. In a murine study it has been shown that bioavailable IGF1 stimulates uteral growth therefore functioning to increase uterine size
. A recent bovine study has suggested that an increase of IGF1 bioavailability has a negative effect on oocyte developmental competence
. An increase in IGF1 gene expression 30 DPP in the present study may infer proliferative effects on the uterus.
SHC2 gene expression was increased 30 DPP and is one of many genes enriching the focal adhesion pathway at this time, suggesting a role for SHC2 in biological processes such as cell differentiation, motility, regulation of gene expression, cell proliferation, and cell survival in the endometrium later in involution. The Src family kinases are non-receptor tyrosine kinases involved in the mediation of intracellular signal transduction to initiate biological processes such as adhesion, migration, invasion, epithelial-to-mesenchymal transition, angiogenesis, apoptosis resistance and proliferation. Src members are activated by the binding of ligands to either their Src homology 2 (SH2) or 3 (SH3) domains
. In bovine endometrial epithelial cells it has been hypothesised that epidermal growth factor receptor may aid in the amplification of oxytocin signalling and activate c-Src resulting in the elevation of prostaglandin F2α production, which is a luteolytic event
Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 14 (SERPINA14) belongs to the serpin superfamily of serine peptidase inhibitors (serpins)
 and has also been previously called uterine milk protein (UTMP). Serine proteases are associated with immune functions involving inflammation, tissue remodelling, pathogen clearance and apoptosis, the over production of which causes pathologies in auto-immune diseases, tumor metastasis and allergies
. Serpins limit the activity of serine proteases thereby regulating the severity of their immune functions
. Gene expression of UTMP has been observed predominantly in the bovine endometrium, ovary and caruncle tissues and the differential allelic expression of which has been associated with longevity in dairy cattle
. Expression of SERPINA14 has been previously shown to be elevated by estrogens during estrus in cattle
[50, 51] and also during pregnancy
. During pregnancy UTMP is thought to have a role in maternal immune modulation, by inhibiting NK-like activity and thus protecting the conceptus in utero. Importantly, another study in sheep suggests that the expression of UTMP in the endometrium is a marker of differentiated and functional glandular epithelium
. In the present study an increase of SERPINA14 expression 30 DPP possibly indicates a greater degree of glandular epithelium repair within the endometrium at this time.
Based on the considerable research performed on these genes in other species, our results point toward an important functional role for SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes, in the restoration of homeostasis during bovine involution.