Bacteria causing pyometra in bitch and queen induce neutrophil extracellular traps.

Neutrophils are capable of releasing their DNA in response to infectious agents to form neutrophil extracellular traps (NETs) to destroy pathogens. Even though pyometra in queens and bitches is a common disease, its pathogenesis is not fully understood. The aim of this study was to assess the presence of NETs in the endometrium of queens and bitches suffering from pyometra. Pyometra and normal uteri were obtained after ovariohysterectomy from adult queens and bitches in diestrus. Uterine contents were evaluated for bacterial isolation and identification and for NETs presence. Escherichia coli were isolated in 5/5 queens and 4/5 bitches, and Streptococcus spp in one bitch. Sterile glass coverslips were placed on the endometrium surface to obtain material for NETs that were evaluated by immunocytochemistry (histone, neutrophil elastase or myeloperoxidase), fluorescence microscopy or scanning electron microscopy. NETs in endometrium content were positively stained by DNA histone DAPI, myeloperoxidase and by neutrophil elastase. NETs were spread in all observed queen and bitch endometria of pyometra cases. Ultrastructure images of NETs depicted clusters of globular material with fine filaments deposited on or around thick filaments and trapped bacteria. To the best of our knowledge these are the first findings confirming NETs endometrial presence in queen and bitch pyometra. Nevertheless, the precise role of NETs in pyometra in the bitch and queen, either to contribute to the defeat of infection or to its persistence remains to be unraveled.

Ovarian steroids, oxytocin, and tumor necrosis factor modulate equine oviduct function.

The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E2), progesterone (P4), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE2 and prostaglandin F2α (PGF2α) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also assessed. During the breeding season, oviduct tissue was obtained post mortem from cyclic mares. Protein of ESR1, ESR2, PGR, AKR1C3, and Ptges was present in OECs, whereas OXTR was shown in oviduct stroma. In follicular phase, protein expression of ESR1, ESR2, PGR, and OXTR increased in oviduct explants (P < 0.05), whereas no estrous cycle effect was noted for AKR1C3 or Ptges. In follicular phase, mRNA transcription was upregulated for Pgr but downregulated for Oxtr, Ptges, and Akr1c3 (P < 0.05). Nevertheless, Esr1 and Esr2 mRNA levels did not change with the estrous cycle. In the ampulla, Esr1, Esr2, and Oxtr mRNA transcription increased, but not for Pgr or Ptges. In contrast, Akr1c3 mRNA level was upregulated in the infundibulum (P < 0.05). In follicular phase, E2, P4, and OXT downregulated PGE2 production by OEC (P < 0.05), but no difference was observed in mid-luteal phase. Explants production of PGE2 rose when treated with OXT in follicular phase; with TNF or OXT in early luteal phase; or with TNF, OXT, or P4 in mid-luteal phase. PGF2α production by OEC was downregulated by all treatments in follicular phase but upregulated in mid-luteal phase (P < 0.05). Oviduct explants PGF2α production was stimulated by TNF or OXT in all estrous cycle phases. In conclusion, this work has shown that ESR1, ESR2, OXTR, Ptges, and AKRLC3 gene transcription and/or translation is estrous cycle dependent and varies with oviduct portion (infundibulum vs ampulla) and cell type. Ovarian steroid hormones, OXT and TNF stimulation of PGF2α and/or PGE2 production is also estrous cycle dependent and varies in the different portions of mare oviduct. Differential transcription level and protein localization in various portions of the oviduct throughout the estrous cycle, as well as PG production, suggest coordinated physiologic actions and mechanisms of steroid hormones, OXT, and TNF in the equine oviduct.

Physiopathologic mechanisms involved in mare endometrosis.

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFß and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1ß, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare.

Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: potent endocrine disruptors inducing infertility.

Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-ß estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E2), progesterone (P4), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study.

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.