Evidence for imbalanced polarization of caruncle macrophages in retained placenta of dairy cows

Retained placenta (RP) is a common reproductive disorder with complex etiology and pathogenesis, affecting approximately 8% of dairy cows during the periparturient period. Macrophages constitute 20% to 25% of all leukocytes at the maternal-fetal interface and coordinate several processes critical for fetal membrane expulsion, including tissue remodeling, induction of apoptosis in damaged cells, and immune activation. This study aimed to investigate the morphological changes at the maternal-fetal interface, as well as the quantity, distribution, and polarization of caruncle macrophages in cows with and without RP. Furthermore, we discuss the potential association between macrophage alterations and histopathological changes in placental tissue of RP cows. A total of 80 Holstein dairy cows (parity 2-4) were enrolled in this study. Blood samples were collected at -7 d before the expected calving date, at calving, at 12 h postpartum, and at 7 d postpartum. Placental tissue samples were collected within 30 min after parturition. Based on whether the placental membranes were expelled within 12 h postpartum, cows were classified retrospectively into normal expulsion (NE; n = 6) and RP (n = 6) groups. Picrosirius red staining, along with elevated mRNA and protein levels of collagen III, indicated enhanced collagen fiber deposition in caruncle tissue. In addition, the mRNA expression of matrix metalloproteinases (MMP2 and MMP9) was downregulated in RP tissues, whereas TIMP1 was upregulated. Compared with NE cows, the apoptosis index and the protein and mRNA levels of pro-apoptotic factors (BAX, Caspase 3, Caspase 8) were lower in cows with RP, and the anti-apoptotic factor (BCL2) was higher, indicating reduced apoptosis in the caruncle tissue from RP cows. In both the serum and tissues, we observed lower levels of chemotactic factors (CXCL1 and MCP-1) in RP cows, alongside increased IL-10 (an immunosuppressive factor) and decreased IL-1β (an immune-stimulatory factor). The downregulated protein and mRNA abundance of the macrophage marker CD68, consistent with reduced presence of CD68⁺ cells observed through immunofluorescence, revealed low numbers of caruncle macrophages in cows with RP. Further, the caruncles tissue of RP cows displayed significant alterations in the distribution of CD68⁺ macrophages, with reduced infiltration into trophoblast cells. Regarding macrophage phenotypic changes in RP cows, the greater protein and mRNA expression of M2 polarization markers (CD206, IL-10, IL-6, and TGF-β), along with greater numbers of CD206⁺/CD68⁺ cells detected through immunofluorescence, indicated that macrophage polarization phenotype in the caruncles of RP cows shifted predominantly toward the M2 phenotype. In contrast, RP cows exhibited lower protein and mRNA levels of M1 polarization markers (CD86, inducible nitric oxide synthase, IL-1β, and NF-κB), as well as reduced numbers of CD86⁺/CD68⁺ cells. Overall, caruncle tissues from RP cows were characterized by a reduced macrophage population with a predominant M2 phenotype. Alterations in the quantity and polarization state of macrophages at the maternal-fetal interface may lead to reduced immune cell trafficking into the caruncle, thus impairing the apoptotic and proteolytic processes essential for placental expulsion.