Characterization of cytokine gene expression in uterine cytobrush samples of non-endometritic versus endometritic postpartum dairy cows

Highlights

• Neutrophil thresholds derived using uterine cytobrush cytology are reflective of the inflammatory state of the bovine uterus.

• Cows with endometritis have a dysregulated expression of pro-inflammatory and regulatory cytokine genes.

• Non-endometritic cows at 29–35 DIM can develop endometritis later in the postpartum period.

• Spontaneous resolution of endometritis can occur during the first two months of postpartum period.

• Spontaneous resolution of endometritis was associated with a lower expression of TGF-β2 gene expression at 29–35 DIM.

Abstract

To better understand uterine inflammation in postpartum dairy cows we collected sequential cytobrush samples at 29–35 and at 49–55 d in milk (DIM). Based on the uterine cytology, cows were classified as Non-endometritic (n = 23; <18% neutrophils) or Endometritic (n = 12; ≥18% neutrophils) at 29–35 DIM and Non-endometritic (n = 17; <10% neutrophils) or Endometritic (n = 9; ≥10% neutrophils) at 49–55 DIM. Cows defined as Sham Controls (n = 6) were examined by vaginoscopy at 29–35 DIM and identified as Non-endometritic (<10% neutrophils) at 49–55 DIM. Cytokine gene expression in cytobrush samples was assessed using qRT-PCR. Sham Controls did not differ significantly (P > 0.17) from Non-endometritic cows at 49–55 DIM and these data were combined (n = 23). Uterine cytology-based classification using the aforementioned thresholds effectively separated cows into groups with Endometritic cows having significantly higher expression of pro-inflammatory (interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17A CSF-1; P < 0.01) and regulatory (IL-1RA and IL-10; P < 0.03) cytokines, relative to Non-endometritic cows. Furthermore, Non-endometritic cows showed a significant decline (P < 0.03) in the expression of pro-inflammatory (IL-1α, IL-6, IL-8) and regulatory (IL-10) cytokine genes as the postpartum period progressed; whereas Endometritic cows exhibited a sustained elevation in transcript abundance throughout the sample period for both pro-inflammatory and regulatory cytokine genes. Expression of transforming growth factor (TGF) genes was more complex with TGF-β3 expression significantly (P < 0.01) lower at 29–35 DIM and TGF-β1 gene expression significantly (P < 0.03) increased at 49–55 DIM in Endometritic versus Non-endometritic cows. Expression of TGF-β2 gene was 2.7-fold higher (P < 0.01) at 29–35 DIM in cows that remained Endometritic when compared to cows recovering by 49–55 DIM. Some Non-endometritic cows (n = 4) at 29–35 DIM were reclassified as Endometritic at 49–55 DIM. The sampling procedures at 29–35 DIM did not alter either the cellular response (P > 0.43) or cytokine gene expression (P > 0.17) at 49–55 DIM. In conclusion, normal uterine involution is characterized by a progressive decline in pro-inflammatory and regulatory cytokine gene expression, while cows with endometritis show a dysregulated inflammatory process characterized by a sustained elevation in pro-inflammatory and regulatory cytokine gene expression. This analysis also shows that decreased TGF-β2 gene expression at 29–35 DIM may be an indicator of recovery from endometritis.

Introduction

Cows must become pregnant, maintain pregnancy, and calve in a timely fashion to maintain the dairy production cycle. How quickly the postpartum uterus returns to its pre-gravid state (uterine involution) and the resumption of normal ovarian cyclicity are two main factors influencing the efficiency of this process. Uterine involution is a complex inflammatory and tissue regenerative process that is completed when the inflammation is resolved, and endometrial repair is complete [[1], [2], [3], [4]]. Diseases, both metabolic and uterine, occurring during the postpartum period can adversely influence normal reproductive events causing subfertility or infertility. Approximately 5–35% of dairy cows experience persistent inflammation of the uterine mucosa known as endometritis caused mainly by opportunistic pathogens such as Escherichia coli and Trueperella pyogenes [2,3]. Endometritis, mainly caused by genital tract commensals [[5], [6], [7], [8]], results in increases in days open, embryonic losses, and calving intervals that translate into reduced productivity for the dairy herd [9,10].

The last two decades of research has focused on the cellular and molecular components of postpartum uterine inflammation and has greatly advanced our understanding of endometritis in cows. Neutrophils are the first and most significant component of the cellular immune response to uterine inflammation [3]. Higher neutrophil proportions (100 X (neutrophils/(leucocytes + endometrial cells))) in uterine cytology samples harvested using either low-volume uterine lavage or uterine cytobrush techniques have become the gold standard for diagnosing endometritis, but the threshold proportion, distinguishing normal involution from true disease, varies among studies [3,11]. Uterine cytology allows us to identify both clinical and subclinical cases of endometritis and to differentiate true uterine disease from conditions that may or may not involve the uterus, such as cervicitis or purulent vaginal discharge. As an indicator of endometritis, a neutrophil threshold of ≥18% has been supported by uterine cytobrush cytology samples harvested during the 4 to 5th wk postpartum [[11], [12], [13]]. As the postpartum period advances the degree of inflammation has been shown to decline as evidenced by lower threshold proportions of neutrophils being indicative of disease [11,12,14].

Gene expression studies using RNA extracted from uterine cytobrush samples have provided useful insights into uterine inflammation. Increased expression of pro-inflammatory cytokine genes, including interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNFα), has been observed in cows diagnosed with postpartum endometritis [12,15]. Released from a variety of cell types, these pro-inflammatory cytokines work as immune messengers to recruit neutrophils and macrophages to the uterine stroma as well as the lumen and promote phagocytosis of bacteria and cellular debris [1,16,17]. The role of other cytokines such as IL-13, IL-17A, IL-18 and colony stimulating factor (CSF)-1 in the endometrium has not been explored. Interleukin 17A, secreted by a number of immune cells, can facilitate neutrophil migration to the site of infection and also induces antimicrobial peptide production by epithelial cells [18]. Originating from the epithelium, CSF-1 is involved in the differentiation, proliferation and maturation of myeloid cells into macrophages [19].

Resolution of an inflammatory process is mediated by regulatory cytokines, such as IL-10, IL-1receptor antagonist (IL-1RA) and the transforming growth factor (TGF)-β family. Increased uterine expression of both IL-1RA gene, a natural non-productive agonist of IL-1 receptor [20], and IL-10 gene [21,22] have previously been reported in cows with endometritis but other studies reported no significant difference in IL-10 gene expression when comparing cows with endometritis versus no uterine disease [15]. The TGF-β cytokine family is produced by epithelial cells, macrophages and NK cells and plays a crucial role in regulating inflammatory damage and tissue development. Transforming growth factor-β gene expression has yet to be reported in cows with endometritis.

Considering the dynamic nature of immune responses at mucosal surfaces and the extensive change in endometrial epithelium during the postpartum period it is crucial to analyze cytokines and chemokines at different time points during the postpartum period to better understand this process. Our main objective was to determine the dynamics of uterine cytokine gene expression in postpartum cows undergoing normal involution versus cows with endometritis. We also investigated whether repeated cytobrush sampling may significantly alter cytokine gene expression or induce an inflammatory response in non-endometritic cows. We hypothesized that distinct, time-dependent changes occur in the expression of both pro-inflammatory and regulatory cytokines during uterine involution and the expression of specific cytokines may be predictive of the persistent inflammatory response associated with endometritis.