CXCR4 signaling at the fetal-maternal interface may drive inflammation and syncytia formation during ovine pregnancy†.

Early pregnancy features complex signaling between fetal trophoblast cells and maternal endometrium directing major peri-implantation events including localized inflammation and remodeling to establish proper placental development. Proinflammatory mediators are important for conceptus attachment, but a more precise understanding of molecular pathways regulating this process is needed to understand how the endometrium becomes receptive to implantation. Both chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal tissues. We identified this pair as a critical driver of placental angiogenesis, but their additional importance to inflammation and trophoblast cell survival, proliferation, and invasion imply a role in syncytia formation at the fetal-maternal microenvironment. We hypothesized that CXCL12 encourages both endometrial inflammation and conceptus attachment during implantation. We employed separate ovine studies to (1) characterize endometrial inflammation during early gestation in the ewe, and (2) establish functional implications of CXCL12 at the fetal-maternal interface through targeted intrauterine infusion of the CXCR4 inhibitor AMD3100. Endometrial tissues were evaluated for inflammatory mediators, intracellular signaling events, endometrial modifications, and trophoblast syncytialization using western blotting and immunohistochemistry. Endometrial tissue from ewes receiving CXCR4 inhibitor demonstrated dysregulated inflammation and reduced AKT and NFKB, paired with elevated autophagic activity compared to control. Immunohistochemical observation revealed an impairment in endometrial surface remodeling and diminished trophoblast syncytialization following localized CXCR4 inhibition. These data suggest CXCL12-CXCR4 regulates endometrial inflammation and remodeling for embryonic implantation, and provide insight regarding mechanisms that, when dysregulated, lead to pregnancy pathologies such as intrauterine growth restriction and preeclampsia.

CXCR4 signaling at the ovine fetal-maternal interface regulates vascularization, CD34+ cell presence, and autophagy in the endometrium†.

Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal-maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.

CXCL12 May Drive Inflammatory Potential in the Ovine Corpus Luteum During Implantation.

Adequate corpus luteum (CL) function is paramount to successful pregnancy. Structural and functional CL integrity is controlled by diverse cell types that contribute and respond to the local cytokine milieu. The chemokine ligand 12 (CXCL12) and receptor, CXCR4, are modulators of inflammation and cell survival, but little is understood about CXCL12-CXCR4 axis and CL functional regulation. Corpora lutea from control nonpregnant ewes (n = 5; day 10 estrous cycle (D10C)) and pregnant ewes (n = 5/day) on days 20 (D20P) and 30 (D30P) post-breeding were analyzed for gene and protein expression of CXCL12, CXCR4, and select inflammatory cytokines. In separate cell culture studies, cytokine production was evaluated following CXCL12 treatment. Abundance of CXCL12 and CXCR4 increased (P < 0.05) in pregnant ewes compared to nonpregnant ewes, as determined by a combination of quantitative PCR, immunoblot, and immunofluorescence microscopy. CXCR4 was detected in steroidogenic and nonsteroidogenic cells in ovine CL, and select pro-inflammatory mediators were greater in CL from pregnant ewes. In vitro studies revealed greater abundance of tumor necrosis factor (TNF) following CXCL12 administration (P = 0.05), while P4 levels in cell media were unchanged. Fully functional CL of pregnant ewes is characterized by increased abundance of inflammatory cytokines which may function in a luteotropic manner. We report concurrent increases in CXCL12, CXCR4, and select inflammatory mediators in ovine CL as early pregnancy progresses. We propose CXCL12 stimulates production of select cytokines, rather than P4 in the CL to assist in CL establishment and survival.

Intrauterine inhibition of chemokine receptor 4 signaling modulates local and systemic inflammation in ovine pregnancy.

PROBLEM: Chemokines help coordinate inflammation within the fetal-maternal microenvironment during gestation. The chemokine CXCL12 signaling through its receptor CXCR4 regulates inflammatory activity, but this phenomenon is not well understood during pregnancy, and there are no reports exploring the role of this pair in peripheral immune tolerance during gestation. Herein, we hypothesize that intrauterine CXCL12-CXCR4 signaling governs local and systemic immunomodulatory dynamics during early gestation in ewes. METHOD OF STUDY: Osmotic pumps were surgically installed for intrauterine infusion of a CXCR4 inhibitor, AMD3100, beginning on day 12 post-breeding in sheep. Endometrial tissues were collected on day 35 of gestation and evaluated for inflammatory potential, Akt pathway activation, and autophagy induction. Demonstrative of peripheral immune activity, levels of select cytokines were assessed in daily blood samples collected throughout the study, as well as in corpus luteum and spleen on day 35. RESULTS: Anti-inflammatory IL10 was primarily localized to endometrial glandular epithelium with lower abundance when CXCR4 was antagonized. Inhibition of CXCR4 at the fetal-maternal interface resulted in less activation of Akt in endometrium, while evidence of autophagy induction was greater. Corpora lutea from ewes receiving intrauterine AMD3100 exhibited lower interferon-gamma (IFNG) expression. Blood inflammatory potential was differentially altered in a temporal fashion throughout infusion. IL10 abundance in spleen was greater following CXCR4 inhibition at the fetal-maternal interface, while IFNG was less. CONCLUSION: Intrauterine CXCL12-CXCR4 signaling governs endometrial and systemic inflammation; disruption of this axis may have detrimental impacts on offspring and maternal health.