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

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.