Prostaglandin D2 is involved in the regulation of inflammatory response in Staphylococcus aureus-infected mice macrophages.

Staphylococcus aureus (S. aureus) is one of the most infamous and widespread bacterial pathogens, causing a hard-to-estimate number of uncomplicated skin infections and probably hundreds of thousands to millions of more severe, invasive infections globally per year. S. aureus may also be acquired from animals, especially in the livestock industry. The interaction mechanism of host and S. aureus has significance for finding ways to against S. aureus infection and control inflammatory response of host, while the molecular biological activities after S. aureus infection, particular in inflammatory and immune cells are not fully clear. The present study aimed to explore whether pattern recognition receptors (PRRs) mediate prostaglandin D2 (PGD2) synthesis and PGD2 participates in the regulation of inflammatory response in macrophages during S. aureus infection or synthetic bacterial lipopeptide (Pam2CSK4) stimulation. PGD2 secretion level was enhanced by mice peritoneal macrophages infected with the S. aureus. The results indicated that PGD2 secretion was impaired in S. aureus infected-macrophages from toll-like receptors 2 (TLR2)-deficient and NLR pyrin domain-containing 3 (NLRP3)-deficient mice. PGD2 synthetase (hematopoietic PGD synthase, HPGDS) inhibitors could reduce the activation of macrophage mitogen-activated protein kinase (MAPK)/nuclear factor-κ-gene binding (NF-κB) signaling pathways. HPGDS inhibition impaired cytokines (TNF-α, IL-1ß, IL-10 and RANTES) secretion and macrophage phagocytosis during S. aureus infection. In addition, inhibition of endogenous PGD2 synthesis was unable to affect the TLR2 and NLRP3 expression in S. aureus-infected macrophages. Taken together, macrophage PGD2 secretion after S. aureus infection depended on receptors TLR2 and NLRP3, and the induced PGD2 participated in the regulation of inflammatory response in S. aureus-infected macrophages. Interestingly, it was found that exogenous PGD2 down-regulated the cytokines secretion and had no effect on phagocytosis in the S. aureus-infected macrophages.

Prostaglandin E2 accumulation is closely associated with S. aureus-infected bovine endometritis.

S. aureus isolated from bacterial bovine endometritis is common in epidemiological reports, but is often ignored as a subclinical pathogenic microorganism. In a previous study, we showed that live S. aureus (LSA) and heat killed S. aureus (HK-SA) induce different inflammatory responses in bovine endometrial tissue, and possibly being associated with the accumulation of prostaglandin E2 (PGE2). Thus, in this study, we varied PGE2 concentrations using inhibitors or agonists in HK-SA-treated bovine endometrial tissues. The results demonstrated that PGE2 has a positive relationship with IL-6, TNF-α, and damage-associated molecular patterns (DAMPs; e.g., HMGB-1 and HABP-1) expression and tissues damage, and is regulated by the EP4-p38 MAPK pathway. We concluded that lipoproteins of S. aureus are associated with PGE2 generation. To further explore the relationship between LSA and PGE2 accumulation, we used the S. aureus strain SA113 lipoprotein knockout (SA113Δlpl) to infect bovine endometrial epithelial cells (BECs). LSA decreased PGE2, cAMP, EP4, IL-6, IL-8, cAMP secretion, and the MAPK and PKA signaling pathways when infected with SA113Δlpl, as compared with SA113-infected groups. Moreover, the adhesion and invasion of BECs were similarly downregulated when lipoproteins in S. aureus were knocked out. The results of this study show that PGE2 is involved in both HK-SA- and LSA-induced inflammatory responses in the bovine endometrium. We suggest that S. aureus infection is associated with bovine endometritis, and although HK-SA and LSA induce different inflammatory responses, the strategy of decreasing PGE2 accumulation is helpful in reducing the inflammation stage caused by S. aureus.

Prostaglandin D2 regulates Escherichia coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages.

Prostaglandin D2 (PGD2) synthesis is closely associated with the innate immune response mediated by pattern recognition receptors (PPRs). We determined PGD2 synthesis whether mediated by Toll-like receptor 2 (TLR2), TLR4 and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) in Escherichia coli (E. coli)-, lipopolysaccharide (LPS)- and Braun lipoprotein (BLP)-stimulated macrophages. Our data demonstrate that TLR2, TLR4, and NLRP3 could regulate the synthesis of PGD2 through cyclo-oxygenase-2 (COX-2) and hematopoietic PGD synthase (H-PGDS) in E. coli-, LPS- or BLP-stimulated macrophages, suggesting that TLR2, TLR4, and NLRP3 are critical in regulating PGD2 secretion by controlling PGD2 synthetase expression in E. coli-, LPS- or BLP-stimulated macrophages. The H-PGDS (a PGD2 specific synthase) inhibitor pre-treatment could down-regulate the secretion of TNF-α, RANTES and IL-10 in LPS- and E. coli-stimulated macrophage. Meanwhile, H-PGDS inhibitor could down-regulate the secretion of TNF-α, while up-regulated RANTES and IL-10 secretion in BLP-stimulated macrophages, suggesting that PGD2 could regulate the secretion of cytokines and chemokines in E. coli-, LPS- or BLP-stimulated macrophages. Furthermore, exogenous PGD2 regulates the secretion of cytokines and chemokines through activation of MAPK and NF-κB signaling pathways after E. coli-, LPS- or BLP stimulation in macrophages. Taken together, PGD2 is found able to regulate E. coli-induced inflammatory responses through TLR2, TLR4, and NLRP3 in macrophages.

Besides TLR2 and TLR4, NLRP3 is also involved in regulating Escherichia coli infection-induced inflammatory responses in mice.

The host Toll-like Receptor-2 (TLR2) and Toll-like Receptor-4 (TLR4) play critical roles in defense against Escherichia coli (E. coli) infection is well-known. The NLR pyrin domain-containing 3 (NLRP3) inflammasome is also an important candidate during the host-recognized pathogen, while the roles of NLRP3 in the host inflammatory response to E. coli infection remains unclear. This study aimed to explore the roles of NLRP3 in regulating the inflammatory response in E. coli infection-induced mice. Our result indicated that compared to wild-type mice, the TLR2-deficient (TLR2-/-), TLR4-deficient (TLR4-/-), and NLRP3-deficient (NLRP3-/-) mice had significant decrease in liver damage after stimulation with Lipopolysaccharide (LPS, 1 µg/mL), Braun lipoprotein (BLP, 1 µg/mL), or infected by WT E. coli (1 × 107 CFU, MOI 5:1). Meanwhile, compared with wild-type mice, the TNF-α and IL-1ß production in serum decreased in TLR2-/-, TLR4-/-, and NLRP3-/- mice after LPS, BLP treatment, or WT E. coli infection. In macrophages from NLRP3-/- mice showed significantly reduced secretion of TNF-α and IL-1ß in response to stimulation with LPS, BLP, or WT E. coli infection compared with macrophages from wild-type mice. These results indicate that besides TLR2 and TLR4, NLRP3 also plays a critical role in host inflammatory responses to defense against E. coli infection, and might provide a therapeutic target in combating disease with bacterium infection.

Braun Lipoprotein Protects against Escherichia coli-Induced Inflammatory Responses and Lethality in Mice.

Escherichia coli (E. coli), a Gram-negative bacterium, is an important pathogen that causes several mammalian diseases. The outer membrane components of E. coli, namely, lipopolysaccharide (LPS) and bacterial lipoprotein, can induce the host innate immune response through pattern recognition receptors (PRRs). However, the detailed roles of the E. coli Braun lipoprotein (BLP) in the regulation of host inflammatory response to E. coli infection remain unclear. In this study, we sought to determine the effects of BLP on E. coli-induced host inflammatory response and lethality using mouse models. Experiments using the E. coli DH5α strain (BLP-positive), E. coli JE5505 strain (BLP-negative), and E. coli JE5505 strain combined with BLP indicated that the presence of BLP could alleviate mortality and organ (liver and lung) damage and decrease proinflammatory cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-1ß [IL-1ß]) and chemokine (regulated on activation normal T-cell expressed and secreted [RANTES]) production in mouse serum and organs. Conversely, E. coli JE5505, E. coli DH5α strain, and E. coli JE5505 combined with BLP treatment induce enhanced anti-inflammatory cytokine (interleukin 10 [IL-10]) production in mouse serum and organs. In addition, BLP could regulate the secretion of proinflammatory cytokines (TNF-α and IL-1ß), chemokines (RANTES), and anti-inflammatory factors (IL-10) through mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling pathways in macrophages. Altogether, our results demonstrate that the bacterial component BLP plays crucial and protective roles in E. coli-infected mice, which may influence the outcome of inflammation in host response to E. coli infection. IMPORTANCE In this study, we investigated the roles of bacterial outer membrane component BLP in regulating inflammatory responses and lethality in mice that were induced by a ubiquitous and serious pathogen, Escherichia coli. BLP could alleviate the mortality of mice and organ damage, as well as decrease proinflammatory cytokines and chemokine production and enhance anti-inflammatory cytokine production in mouse serum and organs. Overall, our results demonstrate that the bacterial component BLP plays crucial and protective roles in E. coli-infected mice through regulating the production of an inflammatory mediator, which may influence the outcome of inflammation in host response to E. coli infection. Our findings provide new information about the basic biology involved in immune responses to E. coli and host-bacterial interactions, which have the potential to translate into novel approaches for the diagnosis and treatment of E. coli-related medical conditions, such as bacteremia and sepsis.

Anti-inflammatory effects of the prostaglandin D2/prostaglandin DP1 receptor and lipocalin-type prostaglandin D2 synthase/prostaglandin D2 pathways in bacteria-induced bovine endometrial tissue.

Dairy cows often develop different degrees of endometritis after calving and this is attributed to pathogenic bacterial infections such as by Escherichia coli and Staphylococcus aureus. Infection of the bovine endometrium causes tissue damage and increases the expression of prostaglandin D2 (PGD2), which exerts anti-inflammatory effects on lung inflammation. However, the roles of PGD2 and its DP1 receptor in endometritis in cows remain unclear. Here, we examined the anti-inflammatory roles of the lipocalin-type prostaglandin D2 synthase (L-PGDS)/PGD2 and DP1 receptor regulatory pathways in bovine endometritis. We evaluated the regulatory effects of PGD2 on inflammation and tissue damage in E. coli- and S. aureus-infected bovine endometrial cells cultured in vitro. We found that the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor (TNF)-α as well as expression of matrix metalloproteinase (MMP)-2, platelet-activating factor receptor (PAFR), and high mobility group box (HMGB)-1 were suppressed after DP1 receptor agonist treatment. In contrast, IL-6, IL-1ß, and TNF-α release and MMP-2, PAFR, and HMGB-1 expression levels were increased after treatment of bovine endometrial tissue with DP1 receptor antagonists. DP1-induced anti-inflammatory effects were dependent on cellular signal transduction. The L-PGDS/PGD2 pathway and DP1 receptor induced anti-inflammatory effects in bovine endometrium infected with S. aureus and E. coli by inhibiting the mitogen-activated protein kinase and nuclear factor-κB signalling pathways, thereby reducing tissue damage. Overall, our findings provide important insights into the pathophysiological roles of PGD2 in bovine endometritis and establish a theoretical basis for applying prostaglandins or non-steroidal anti-inflammatory drugs for treating endometrial inflammatory infertility in bovines.

Prostaglandin E receptor 2 mediates the inducible effects of prostaglandin E2 on expression of growth factors and enzymes in cattle endometrial epithelial cells and explants.

Prostaglandin E2 (PGE2 ) is able to induce the expression of several growth factors and enzymes in cattle endometria. However, the specific type of PGE2 receptors which mediates this effect is not fully clear. In this study, the role of prostaglandin E receptor 2 (PTGER2) in PGE2 -mediated induction of growth factors and enzymes expression in cattle endometrial explants and epithelial cells were investigated. PTGER2 was blocked by a PTGER2 antagonist, AH6809, before PGE2 treatment, then the mRNA and protein expression levels of several growth factors and enzymes were compared with that in PGE2 alone treatment group by real-time RT-PCR and Western blotting analysis in endometrial epithelial cells and explants. Results indicated that PGE2 significantly increased the mRNA and protein levels of these growth factors and enzymes, while the rates of increment in the expression of these growth factors and enzymes were inhibited by AH6809. In addition, a PTGER2 agonist, butaprost, significantly increased the expression levels of these growth factors and enzymes, and the effect could be blocked by AH6809. In conclusion, PTGER2 was found to be one dominant receptor mediating the inducible effects of PGE2 on the expression of these growth factors and enzymes in cattle endometrial explants and epithelial cells.

TLR2, TLR4, and NLRP3 mediated the balance between host immune-driven resistance and tolerance in Staphylococcus aureus-infected mice.

Staphylococcus aureus (S. aureus) is a gram-positive pathogen that can cause infectious diseases in mammals. S. aureus-induced host innate immune responses have a relationship with Toll-like receptor 2 (TLR2), TLR4, and Nod-like receptor pyrin domain-containing protein 3 (NLRP3). However, the detailed roles of TLR2, TLR4, and NLRP3 in regulating the host inflammatory response to S. aureus infection remain unclear. Our data indicated that the S. aureus-induced mortality was aggravated by deficiency of TLR2, TLR4, and NLRP3 in mice. In the subsequent experiment, we found that during S. aureus infection, the roles of TLR2, TLR4, and NLRP3 seemed to be different at multiple timepoints. The deficiency of TLR2, TLR4, or NLRP3 attenuated the expression of High-mobility group box protein 1 (HMGB1) and Hyaluronic acid-binding protein 2 (HABP2), which is accompanied by decreased proinflammatory cytokine (TNF-α), chemokine (RANTES), and anti-inflammatory cytokine (IL-10) production in lungs and serum at 3 h and 6 h post-infection. However, with S. aureus infection prolonged (24 h post-infection), the trend was diametrically opposite. The results showed that deficiency of TLR2, TLR4, or NLRP3 aggravated HABP2 and HMGB1 expression, which is accompanied by enhanced proinflammatory cytokine (TNF-α), chemokine (RANTES), and anti-inflammatory cytokine (IL-10) production in lungs and serum. These results were consistent with the data observed in S. aureus-infected bone marrow-derived macrophages (BMDMs). All these results suggested that during S. aureus infection, TLR2, TLR4, and NLRP3 has time-dependent effect in regulating the balance between immune-driven resistance and tolerance.

Codonopsis pilosula polysaccharides attenuate Escherichia coli-induced acute lung injury in mice.

Acute lung injury (ALI) is an inflammatory lung disease that is caused by bacterial infection. Lipopolysaccharide (LPS), a prototype pathogen-associated molecular pattern (PAMP) from Gram-negative bacteria such as Escherichia coli (E. coli), is an essential risk factor for ALI. LPS and E. coli induced the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-κB) signaling pathways, which led to the increasing immune molecule transcription, including pro-inflammatory cytokine and chemokine secretion. Codonopsis pilosula polysaccharides (CPPS) exhibit various biological activities and pharmacological effects. However, the effect of CPPS on ALI caused by LPS stimulation or E. coli infection remains unclear. Our results showed that CPPS (6.25, 12.5, 25, or 50 µg mL-1) could attenuate the secretion of TNF-α and IL-1ß and impair the phosphorylation of ERK, p38 and p65 in E. coli-infected macrophages without causing toxic reactions. In addition to regulating the secretion of pro-inflammatory cytokines and the activation of MAPK and NF-κB signaling pathways, CPPS could enhance bacterial phagocytosis and intracellular killing in macrophages, and inhibit the bacterial growth of E. coli. In vivo experiments showed that CPPS attenuated LPS- and E. coli-induced lung damage in mice, which was characterized by decreased pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) and chemokine (RANTES) production and production of the biomarkers of tissue damage (HABP2 and HMGB1) in the lungs. Altogether, this study demonstrated that CPPS have a protective effect on the lungs in LPS- and E. coli-induced ALI mouse models, suggesting that CPPS could be a potential drug for the treatment of ALI.

Proliferation of bovine endometrial epithelial cells is promoted by prostaglandin E2-PTGER2 signaling through cell cycle regulation.

It is known that prostaglandin E2 (PGE2) induces proliferation of epithelia in bovine endometrial explants, however, the detailed mechanism of regulation of PGE2 in inducing bovine endometrial epithelial cell (bEEC) proliferation is unclear. In this study, we determined whether proliferation of bEECs is promoted by PGE2-prostaglandin E receptor 2 (PTGER2) signaling activation through cell cycle regulation. The results demonstrated that bEECs proliferation was induced by treatment of PGE2 and PTGER2 agonist butaprost. These processes were down-regulated by PTGER2 antagonist AH6809 and CDK inhibitors (LEE011, CDK2 Inhibitor II and Ro 3306). PGE2 and butaprost induced cyclins (A, B1, D1, D3 and E2), cyclin-dependent kinases (CDKs, 1, 2, 4 and 6), and epidermal growth factor (EGF) expression were inhibited by AH6809 treatment in bEECs. Moreover, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and PTGER2 expression in bEECs were up-regulated by PGE2 and butaprost treatment. Our data demonstrate that PGE2-PTGER2 signaling activation has a direct molecular association with cell cycle regulation and cell proliferation in bEECs. Collectively, these findings will improve our understanding of the roles for PGE2-PTGER2 signaling activation in the physiological and pharmacological processes of bovine endometrium.

Live S. aureus and heat-killed S. aureus induce different inflammation-associated factors in bovine endometrial tissue in vitro.

Staphylococcus aureus is majorly involved in bovine mastitis; however, it weakly induces pro-inflammatory factors in mammary gland epithelial cells. We aimed to clarify the involvement of S. aureus in other inflammation types and its relationship with inflammatory factor secretion in bovine endometritis. We used live S. aureus (LSA)- and heat-killed S. aureus (HK-SA)-treated bovine endometrial tissue in vitro. The HK-SA-treated group showed significantly higher IL-6, IL-1ß, TNF-α, CXCL1/2 and TLR2 expression than the LSA-infected group. Contrastingly, the LSA-infected group showed significantly higher PTGS2, mPGES-1, and EP4 expression than the HK-SA treated group. There was no significant between-group difference in hyaluronan-binding protein 1 expression, which suggested similar inflammatory responses. H&E results indicated that LSA and HK-SA induced shedding of endometrial gland epithelial cells. The LSA-infected group showed higher high-mobility group box 1 protein expression than the HK-SA treated groups, which indicated differences in signaling pathway activation. Further, the LSA-treated group had higher JNK and p38 MAPK levels while the HK-SA-treated group had higher IκB-α levels. There was no significant between-group difference in the ERK signaling pathway. Our findings indicate that the pathogen-associated molecular patterns (PAMPs) of S. aureus activate pro-inflammatory factor expression via the TLR2-ERK-NF-κB signaling pathway. Contrastingly, LSA induced PGE2 accumulation via the TLR2/MAPKs signaling pathway. This is the first report that S. aureus and the PAMPs of S. aureus activate different signaling pathways and that LSA mainly induce PGE2 accumulation rather than cytokine secretion.

Prostaglandin E2 promotes Staphylococcus aureus infection via EP4 receptor in bovine endometrium.

Prostaglandin E2 (PGE2) enhances Staphylococcus aureus infection but its mechanism is not well understood. Here, we examined the effect of PGE2 on Staphylococcal Protein A (SPA) expression in bovine endometrium and determined the role of select PGE2 receptors (i.e., EP2 and EP4) in adhesion and internalization of S. aureus. S. aureus isolate SA113 was used for in vitro infection of bovine endometrial tissues and epithelial cells, with treatment conditions consisting of untreated control, SA113 treatment, SA113 + PGE2, SA113 + PGE2 + EP2 receptor antagonist (AH-6809), and SA113 + PGE2 + EP4 receptor antagonist (AH-23848). Immunofluorescence assay revealed that PGE2 could promote SPA expression in S. aureus-infected bovine endometrial tissues. PGE2 also enhanced the adhesion and internalization of S. aureus in bovine endometrial cells. The addition of EP4 antagonist, but not the EP2 antagonist, abrogated the ability of PGE2 to promote S. aureus SPA expression, adhesion, and internalization in endometrial cells. Our findings suggest that S. aureus infection in the endometrium is enhanced by PGE2 through the EP4 receptor. This result is essential for the development of new approach to treating S. aureus infection, such as the application of EP4 antagonist as an adjunct drug treatment.

Lipopolysaccharide stimulates bovine endometrium explants through toll­like receptor 4 signaling and PGE2 synthesis.

Bovine endometrium infection with gram-negative bacteria commonly causes uterine diseases. Previous studies indicate that prostaglandin E2 (PGE2) is an inflammatory mediator in bacterial endometritis. However, the mechanism underlying lipopolysaccharide (LPS)-induced inflammatory response regulation in bovine endometrial explants remains elusive. In the present study, bovine explants were pre-treated with 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitors before LPS stimulation. PGE2 secretion, prostaglandin synthetase, pro-inflammatory factor, damage-associated molecular pattern (DAMP), and related signaling pathway factor levels were evaluated. Using 15-PGDH inhibitors pre-treatment, LPS-treated bovine endometrial explants exhibited augmentation of PGE2 and DAMP expression, and upregulation of various signaling pathway factors. Protein kinase A (PKA), extracellular-signal-regulated kinase, and c-Jun N-terminal kinase phosphorylation and degradation of nuclear transcription factor-κB (NF-κB) inhibitors were induced in the pre-treated endometrial explants. The mechanism underlying LPS-induced PGE2 accumulation acting as a pro-inflammatory mediator through toll-like receptor 4 signaling in bovine explants could involve the PKA, mitogen-activated protein kinase, and NF-κB pathways.

TLR2/4 promotes PGE2 production to increase tissue damage in Escherichia coli-infected bovine endometrial explants via MyD88/p38 MAPK pathway.

Prostaglandin E2 (PGE2), a lipid mediator, is released by several cell types including endometrial cells and plays a central role in bacterial infection of the endometrium during inflammation. PGE2 production accumulated in Escherichia coli (E. coli) -infected bovine endometrial tissue, which increased E. coli-infected endometrial tissue damage. However, the mechanisms of PGE2 accumulation in the E. coli-infected endometrium during inflammation-associated endometrial tissue damage remain unclear. This study was conducted to investigate the role of Toll-like receptors (TLRs) 2 and 4 in increased PGE2 production in E. coli-infected endometrial tissue. E. coli and TLR2/4 agonists significantly induced cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression and PGE2 synthesis detected by RT-PCR, Western blot, and ELISA in the endometrial tissue. The expression and synthesis were dramatically decreased by TLR4, myeloid differentiation factor88 (MyD88), and p38 mitogen-activated protein kinase (MAPK) inhibitors in E. coli-infected endometrial tissue. These inhibitors also significantly decreased proinflammatory factor (interleukin-6 and tumor necrosis factor-α) and damage-associated molecular pattern (high mobility group box-1 and hyaluronan-binding protein-1) release and tissue damage measured by double-label immunofluorescence in E. coli-infected endometrial explants. Our work provides in vitro evidence that TLR2/4-MyD88/p38 MAPK promotes PGE2 synthesis and E. coli-infected endometrial tissue damage, which may be useful for improving PGE2-based therapies for endometritis.

Developing a 3D animation tool to improve veterinary undergraduate understanding of obstetrical problems in horses.

BACKGROUND: Many challenges are encountered in both teaching and learning veterinary obstetrics. This may be due to outdated teaching materials, as the main model of content transmission remains centred around text and images. METHODS: Visualisation methods such as three-dimensional (3D) and Graphics Interchange Format (GIF) tools were applied in an attempt to improve obstetrics education outcomes in the third-year class. Traditional teaching methods were utilised in the fourth-year and fifth-year students. RESULTS: These supplementary tools significantly increased the third-year students' final examination results compared with the results of fourth-year and fifth-year students (P<0.05). These examinations were designed to evaluate comprehension of the subject matter. Self-assessment questionnaire results further indicated that 3D animation and GIF promoted learning efficiency. CONCLUSION: Incorporation of 3D animation learning tools into the veterinary curriculum is predicted to better prepare students for the management of obstetrical cases after graduation.

17ß-estradiol regulates prostaglandin E2 and F2α synthesis and function in endometrial explants of cattle.

Prostaglandins (PG) have primary functions in the reproductive tract, however, the mechanism of regulation of PG secretion in the endometrium is unclear. Estrogen as a predominant regulator of uterine functions during the mammalian estrous cycle and effects of estrogen on synthesis of PG and function in uterine tissues of cattle are not fully understood. In this study, there was evaluation of the concentration- and time-effects of 17ß-estradiol on PG synthesis in endometrial explants of cattle, focusing on the secretion of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) as well as relative abundance of mRNA transcript and protein for both the enzymes responsible for PGE2 and PGF2α synthesis, including prostaglandin-endoperoxide synthase 1 and 2 (PTGS1, PTGS2), PGE2 synthase (PGES), PGF2α synthase (PGFS), and carbonyl reductase (CBR1), and the receptors responsible for downstream PGE2 (PTGER2, PTGER4) and PGF2α (PTGFR) signaling. Results indicated that 17ß-estradiol increased PGE2 and PGF2α production at concentrations ranging from 10-11 to 10-8 M. Furthermore, abundances of PTGS1, PTGS2, PGES, PGFS, PTGER2, PTGER4, and PTGFR mRNA transcripts and protein were greater immediately after 17ß-estradiol treatment at almost all the concentrations, while these CBR1 abundances were less as a result of treatments with 17ß-estradiol. These data support the hypothesis that estradiol modulates the synthesis and function of PG in the endometrium of cattle.

Prostaglandin F2α-PTGFR signaling promotes proliferation of endometrial epithelial cells of cattle through cell cycle regulation.

There is production of prostaglandin F2α (PGF2α) and there is PGF2α receptor (PTGFR) mRNA transcript in endometrial epithelial cells of cattle. The aims of the present study were to (1) determine whether PGF2α-PTGFR signaling modulates the proliferation of endometrial epithelial cells and (2) increase knowledge of PGF2α-PTGFR signaling on the physiological and pharmacological processes in the endometrium of cattle. Amount of cellular proliferation was determined using real-time cell analysis and cell proliferation reagent WST-1 procedures. Abundance of cyclins, cyclin-dependent kinases (CDKs), cyclin-kinase inhibitors, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), PTGFR, epidermal growth factor (EGF) mRNA and protein abundances were evaluated using real-time RT-PCR and western blot analyses. The PGF2α-PTGFR signaling promoted the proliferation of endometrial epithelial cells by inducing changes in abundance of mRNA transcript and protein that resulted in an increase in the abundance for the cyclins (A, B1, D1, D3), CDKs (1, 2, 4, 6), and PCNA; decrease in abundance for p21; and increase in abundance for EGF, COX-1, COX-2, and PTGFR. There was a direct molecular association between PGF2α-PTGFR signaling and cell cycle regulation in endometrial epithelial cells of cattle. In addition, findings improve the understanding of PGF2α-PTGFR signaling in the physiological and pharmacological processes of the endometrium of cattle.

EP2/4 Receptors Promote the Synthesis of PGE2 Increasing Tissue Damage in Bovine Endometrial Explants Induced by Escherichia coli.

The bovine uterine is easily contaminated with bacteria during coitus or parturition. A previous study suggested that prostaglandin E2 (PGE2) promoted Escherichia coli-infected bovine endometrial tissue inflammatory damage via cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1). However, it remains unclear which PGE2 receptors regulate the proinflammatory effect of PGE2 In this study, we evaluated the effect of PGE2 and its mediated receptors on E. coli-infected endometrium explants isolated from the bovine uterus. The E. coli-infected bovine endometrial explants were cultured in vitro, and the study used EP2/4 receptor agonists to investigate the responses of COX-2, mPGES-1, PGE2, proinflammatory factors, and damage-associated molecular patterns (DAMPs). The expression of COX-2, mPGES-1, PGE2, proinflammatory factors, and DAMPs was significantly increased after infection with E. coli; however, the high expression levels caused by E. coli were reduced following treatment with COX-2 and mPGES-1 inhibitors. In addition, the expression levels of COX-2, mPGES-1, PGE2, proinflammatory factors, and DAMPs were higher in treatment with EP2/4 receptor agonists in E. coli-infected endometrium explants, and their promotable effects were effectively blocked by EP2/4 receptor antagonists. These findings provide evidence that PGE2 may promote the progress of inflammation in endometrial explants infected with E. coli in bovines. Furthermore, EP2/4 may be involved in a positive feedback loop for COX-2 and mPGES-1 expression, and this may be responsible for the proinflammatory reaction of PGE2 in E. coli-infected uteri of bovines. SIGNIFICANCE STATEMENT: PGE2 promoted E. coli-infected bovine endometrial tissue damage via COX-2 and mPGES-1. However, this proinflammatory effect of PGE2 depends on which receptors are affected by PGE2, and this remains unclear. In this study, it was investigated that EP2 and EP4 may be involved in a positive feedback loop for COX-2 and mPGES-1 expression, and this may be responsible for the proinflammatory reaction of PGE2 in E. coli-infected uteri of bovines.

Prostaglandin E2 promotes nitric oxide synthase 2, platelet-activating factor receptor, and matrix metalloproteinase-2 expression in Escherichia coli-challenged ex vivo endometrial explants via the prostaglandin E2 receptor 4/protein kinase a signaling pathway.

Prostaglandin E2 (PGE2) is an inflammatory mediator involved in the pathogenesis of several chronic inflammatory conditions, including endometritis. Previous studies have shown that PGE2 accumulates in Escherichia coli-challenged ex vivo endometrial explants, increasing the expression of pro-inflammatory factors and aggravating tissue damage; these alterations are linked to key enzymes involved in the synthesis of PGE2, including cyclooxygenases-2 (COX-2) and microsomal PGES-1 (mPGES-1). In this study, we aimed to investigate whether administration of PGE2 modulated the activities of nitric oxide synthase 2 (NOS2), platelet-activating factor receptor (PAFR), and matrix metalloproteinase (MMP)-2 in E. coli-challenged ex vivo bovine endometrial explants. Our findings showed that COX-2 and mPGES-1 inhibitors significantly reduced NOS2, PAFR, and MMP-2 expression in the E. coli-challenged ex vivo endometrial explants. In addition, NOS2, PAFR, and MMP-2 expression levels were strongly increased in response to treatment with 15-prostaglandin dehydrogenase inhibitors in the E. coli-challenged ex vivo endometrial explants. However, these stimulatory effects could be blocked by PGE2 receptor 4 (EP4) and protein kinase A (PKA) inhibitors. Overall, these findings show that pathogenic PGE2 upregulated NOS2, PAFR, and MMP-2 expression, which may enhance inflammatory damage via the EP4/PKA signaling pathway in E. coli-challenged ex vivo endometrial explants.

Regulatory roles of PGE2 in LPS-induced tissue damage in bovine endometrial explants.

Bovine endometritis is the most common uterine disease following parturition. The role of prostaglandin E2 (PGE2) in regulating normal physiological function in the bovine endometrium has been clearly established. Although PGE2 accumulation is observed in multiple inflammatory diseases, such as endometritis, its association with pathogen-induced inflammatory damage in the endometrium is unclear. To clarify the role of PGE2 in lipopolysaccharide (LPS)-induced endometritis in cultured bovine endometrial explants, the levels of PGE2 secretion, prostaglandin synthetases, pro-inflammatory factors, and damage-associated molecular patterns (DAMPs) were evaluated in the present study. Significant PGE2 accumulation in response to LPS stimulation, up-regulation of prostaglandin-endoperoxide synthase-2 (PTGS-2), microsomal prostaglandin E synthase-1 (mPGES-1), pro-inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and induced nitric oxide synthase (iNOS)/nitric oxide (NO) and DAMPs including hyaluronan binding protein 1 (HABP1) and high mobility group box-1 (HMGB1), were observed compared to the control group. LPS induced distinct damage in the bovine endometrium, characterized by morphological changes and increases in HABP1 and HMGB1 expression. PTGS-2 inhibitors CAY10404 and NS398 effectively decreased the secretion of PGE2 and the expression of prostaglandin synthetases, pro-inflammatory factors and DAMPs, and alleviated LPS-induced tissue damage. These results indicate that PGE2 accumulates via PTGS-2 and mPGES-1 and induces tissue damage by upregulating pro-inflammatory factors and DAMPs in LPS-treated bovine endometrial explants. These findings provide a basis for the effect of PGE2 on LPS-treated bovine endometrium, and suggest a potential target for curing endometritis.