Pro- and anti-inflammatory mediators change leukotriene B4 and leukotriene C4 synthesis and secretion in an inflamed porcine endometrium

Domestic Animal Endocrinology xxx (2014) 1–11

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1 2 3 4 4 4 5 6 Q1 7 8 Q 10 J. Czarzasta, A. Andronowska, B. Jana* 9 Division of Reproductive Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-748 Olsztyn, 10 Q 2 Poland 11 12 13 a r t i c l e i n f o a b s t r a c t 14 15 Article history: We studied the effect of lipopolysaccharide (LPS), proinflammatory cytokines (tumor neReceived 13 February 2014 16 crosis factor a [TNF-a] and interleukin [IL] 1b), and anti-inflammatory cytokines (IL-4 and Received in revised form 13 May 2014 17 IL-10) on leukotriene (LT) A4 hydrolase and LTC4 synthase (LTCS) protein expression in, and Accepted 14 May 2014 LTB4 and LTC4 secretion from, an inflamed porcine endometrium. On day 3 of the estrous 18 cycle (day 0 of the study), 50 mL of either saline or Escherichia coli suspension 19 Keywords: (109 CFU/mL) was injected into each uterine horn of gilts (n ¼ 12 per group). Endometrial 20 Uterine inflammation explants, obtained 8 and 16 days later, were incubated for 24 h with LPS (10 or 100 ng/mL 21 Leukotriene of medium), TNF-a, IL-1b, IL-4, and IL-10 (each cytokine: 1 or 10 ng/mL of medium). Cytokines 22 Although acute endometritis developed in all bacteria-inoculated gilts, a severe form of Lipopolysaccharide 23 acute endometritis was diagnosed more often on day 8 of the study than on day 16. The Gilt 24 amount of the leukotriene A4 hydrolase protein in the inflamed endometrium on day 8 was 25 greater after applying the lower dose of TNF-a (P < 0.001) and both doses of IL-1b (P < 26 0.001) and IL-4 (1 ng, P < 0.01 and 10 ng, P < 0.001) than in the saline-treated uteri. A 27 similar situation was observed in the case of the inflamed tissue on day 16 in response to 28 LPS (100 ng, P < 0.01), TNF-a (10 ng, P < 0.05), and IL-4 (1 ng, P < 0.001). The content of LTC4 synthase in the inflamed endometrium on day 8 was reduced by LPS (100 ng, P < 29 0.05), IL-1b (10 ng, P < 0.05), IL-4 (1 and 10 ng, P < 0.05), and IL-10 (1 ng, P < 0.01) but 30 increased after the application of LPS (100 ng, P < 0.05) and TNF-a (P < 0.001), IL-1b, and 31 IL-4 (1 ng, P < 0.05 and 10 ng, P < 0.001) on day 16. On day 8, endometrial secretion of LTB4 32 from the saline-injected and E coli-injected organs was similar in response to all of the 33 used mediators. On the other hand, the contents of LTB4 in the medium decreased after 34 incubating the inflamed tissues from day 16 with TNF-a (1 ng, P < 0.05 and 10 ng, P < 35 0.01), IL-1b (1 ng, P < 0.01), and IL-10 (10 ng, P < 0.05) compared with the saline-treated 36 ones. Secretion of LTC4 from the inflamed uteri on day 8 was elevated by the lower doses of 37 TNF-a (P < 0.01) and IL-10 (P < 0.05), whereas on day 16, such an effect occurred in 38 response to the higher doses of IL-4 (P < 0.01) and IL-10 (P < 0.05). The obtained results show that pro- and anti-inflammatory mediators participate in the synthesis/secretion of 39 LTs from an inflamed porcine endometrium. Our data suggest that inflammatory mediators 40 may indirectly affect the processes regulated by LTs by influencing LT production. 41 Ó 2014 Elsevier Inc. All rights reserved. 42 43 44 45 1. Introduction 46 47 Uterine inflammation (endometritis/metritis) is the * Corresponding author. Tel.: þ48 89 53574 37; fax: þ48 89 5357421. 48 most frequent reproductive disorder in livestock. The E-mail address: [email protected] (B. Jana). 49

Pro- and anti-inflammatory mediators change leukotriene B and leukotriene C synthesis and secretion in an inflamed porcine endometrium

0739-7240/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2014.05.001

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consequences of uterine infections are manifold and can range from a complete lack of influence on reproductive performance to perpetual infertility. The most common causes of removing pigs affected by endometritis from herds are abnormal vaginal discharge, no pregnancy, and anestrus [1]. The uteri of sows suffering from endometritis and those free of the condition are a source of bacteria such as Escherichia coli, Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Corynebacterium spp. [1–3]. Uterine inflammation causes considerable alterations in the synthesis and secretion of PGF2a, PGE2 [4,5], PGI2 [6], thromboxane A2 [7], and nitric oxide [8]. Leukotrienes (LTs) are a family of potent lipid messengers that play a role in the innate immune and inflammatory responses. Their production takes place mainly in immunologic cells. The synthesis of LTs is initiated by the release of arachidonic acid from the cell membrane influenced by phospholipase A2. Subsequently, arachidonic acid is converted in a two-step process into an unstable intermediate, LTA4, by a catalytic complex of 5lipoxygenase (5-LO) and the 5-LO–activating protein. This substance is further transformed by cytosolic LTA4 hydrolase (LTAH) to LTB4 or generated to tripeptide glutathione by LTC4 synthase (LTCS) to give LTC4. LTC4 and its metabolites, LTD4, and LTE4 are collectively known as cysteinyl-LTs [9–11]. Earlier studies have shown that in immunologic cells, interleukin (IL) 4 and IL-13 stimulate the expression of 5LO, LTAH, and LTCS [12,13], whereas lipopolysaccharide (LPS) augments the production of LTB4 [14]. In turn, the tumor necrosis factor a (TNF-a) is able to enhance 5-LO messenger RNA level in the intestinal epithelium [15], whereas IL-1b and IL-6 promote the release of an LO product, 5-hydroxyeicosatetraenoic acid, from human gestational tissues [16]. Conversely, an inhibitory effect of IL-4 and IL-13 on LTB4 release from human peripheral monocytes has been noted [17]. Large quantities of TNF-a, IL-1, IL-6, IL-8, and IL-10 are known to be synthesized within and secreted from the uterus during inflammation [18–22]. Moreover, our previous studies revealed that the inflammation of the uterus in gilts was connected with an increase in the contents of LTB4 and LTC4 in peripheral blood and uterine tissues and washings, which was in turn associated with the enhanced expression of 5-LO, LTAH, and LTCS [23]. Additionally, under physiological conditions, functional receptors for TNF-a and IL-1b were found in the bovine [24] and porcine [25] endometrium, respectively, and ones for IL-4 in human uteroplacental tissues [26]. The expression of IL-10 receptors was revealed in human adenomyotic tissues [27]. Based on the previously mentioned findings, we hypothesize that pro- and anti-inflammatory factors may indirectly affect processes regulated by LTs by modulating LT synthesis/secretion in an inflamed uterus. Because the mechanisms involved in controlling the production and release of LTs in an inflamed uterus are not completely recognized, the present study was conducted to determine the influence of LPS and proinflammatory (TNFa and IL-1b) and anti-inflammatory (IL-4 and IL-10) cytokines on (1) the amounts of LTCS and LTAH proteins in an inflamed porcine endometrium and (2) the release of LTB4 and LTC4 from this tissue.

2. Materials and methods 2.1. Animals and experimental procedures All animals were kept and treated in accordance with procedures which had the consent of the local ethics committee (conforming to the principles of animal care, NIH publication No 86–23, revised in 1985, Agreement No 31/2010). The study was performed on 30 gilts (Large White  Landrace) aged 7 to 8 mo and weighing between 107 and 122 kg. Behavioral estrus was determined with the aid of a tester boar. The animals originated from a herd which was characterized by no disturbances in reproductive processes. The experimental procedure concerning the induction of endometritis had been described earlier in greater detail [6]. To provide a brief summary of the method, 24 pigs were randomly chosen and designated into either a salinetreated group (SAL, n ¼ 12) or E coli-treated group (E coli, n ¼ 12) on day 3 of the estrous cycle (day 0 of the experiment). Next, the animals were subjected to mid-ventral laparotomy under general anesthesia, and either 50 mL of saline solution or 50 mL of E coli suspension (strain O25:K23/a/:H1; Department of Microbiology, National Veterinary Research Institute, Pu1awy, Poland), containing 109 CFU/mL, was administered into each uterine horn. The first subset of the saline-treated animals (n ¼ 6) and E colitreated animals (n ¼ 6) was slaughtered on day 8 and the second on day 16 after treatment (expected days 11 and 19 of the estrous cycle), at which time the uteri were collected. In addition to this, the uteri of pigs from the control group (CON, n ¼ 6, not subjected to any investigative procedures) were collected on day 0 (day 3 of the estrous cycle) to determine the basal values of the tested variables. The results of macroscopic and histopathologic examination of uteri used in the present study had been reported in an earlier work [6]. 2.2. Treatment of endometrial fragments To conduct in vitro studies, the uteri were transported on ice to the laboratory within 30 min and then washed twice with sterile phosphate-buffered saline (137 mM NaCl, 27 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4; pH 7.4). Next, a fragment of the uterine wall was taken from the middle part of each horn, and the endometrium was separated from the myometrium by careful scraping, using a scalpel blade. Subsequently, the fragments of the endometrium were cut and sliced (100–110 mg) and washed with Medium 199 (Sigma, No. A2058). Single explants of the endometrium were placed into glass vials containing 2 mL of Medium 199 supplemented with 0.1% BSA (Sigma, No. Q 4 A2058) and antibiotics (100 IU/mL of penicillin and 10 mg/ mL of streptomycin, both from Sigma). The tissue fragments were preincubated and incubated in a shaking water bath at 37 C in a humidified atmosphere of 95% air and 5% CO2. After a 90-min preincubation period, the endometrial explants were treated for 24 h with fresh (control) medium or with the addition of 10 and 100 ng/mL of LPS (Sigma, No. L2880) and 1 and 10 ng/mL of TNF-a (Sigma, No. T6674), IL-1b (Sigma, No. SRP3083), IL-4 (Sigma, No. SRP4137), and

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IL-10 (Sigma, No. SRP3071). Moreover, to determine the reactivity of the endometrial tissue, the amount of PGF2a in the medium was estimated after the use of 104 M of nitric oxide donor (NONOate; Cayman Chemical Co, No. 82150) [28]. Initial dilutions of the used agents were done according to the manufacturer’s instructions (ie, LPS, TNF-a, IL-1b, and IL-4 were diluted in 0.2 mm-filtrated distilled water, IL-10 in 5 mM Na3PO4 [pH 7.2], and NONOate in 0.01 M NaOH) and then stored at 20 C. The final solutions of these agents were prepared using the same medium as for tissue preincubation and incubation. All treatments were performed in triplicates for each of the six gilts in the individual groups. The doses of TNF-a and IL-1b used in the experiment were based on the results of Jana et al [29]. In turn, the doses of LPS, IL-4, IL-10, and NONOate and the incubation time were assessed in the preliminary study. After incubation, the endometrial fragments were stored at 80 C until the content LTAH and LTCS proteins were analyzed. The medium was placed into tubes containing 10 mL of stabilizing mixture (0.3 M EDTA, POCH Gliwice, Poland; 1% aspirin; Polfa, Starogard, Poland) and stored at 20 C for further analysis of LTB4 and LTC4 concentrations. 2.3. Western blot procedure The expression of LTAH and LTCS proteins in the endometrial explants was determined in accordance with an earlier described procedure [29]. Briefly, the tissue fragments were homogenized on ice with a cold buffer (50 mM Tris–HCl, pH 7.4; 10 mM EDTA, 150 mM NaCl, 1% Triton X100 with 1 mM pepstatin A, 5 mg/mL of leupeptin, 5 mg/mL of aprotinin, and 1 mM phenylmethylsulfonyl fluoride; all reagents purchased from Sigma) and centrifuged (10 min at 2500 g, 4 C). The supernatants were centrifuged (1 h at 17, 500 g, 4 C), and pellets were stored at 80 C for further analysis. The method by Bradford [30] was used to estimate protein levels. Equal amounts (20 mg) of tissue lysates were dissolved in sodium dodecyl sulfate, a gel-loading buffer, heated (95 C, 4 min), and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The separated proteins were then electroblotted onto 0.45-mm nitrocellulose membranes in a transfer buffer. The nonspecific binding sites were blocked by incubation with 5% fat-free dry milk in Tris-buffered saline Tween 20 buffer at room temperature for 1.5 h. The nitrocellulose membranes were incubated overnight at 4 C with primary antibodies, including rabbit antihuman LTAH polyclonal antibody (diluted 1:250; Cayman Chemical Co, No. 160250) and rabbit antihuman LTCS polyclonal antibody (diluted 1:250; Santa Cruz Biotechnology, No. sc 20,108). LTAH and LTCS were detected by incubating the nitrocellulose membranes with secondary biotinylated goat anti-rabbit antibody for 1.5 h (room temperature; diluted 1:3,000; Vectastain ABC kit; Vector Laboratories, No. PK4001). Visualization of the immune complexes was done by incubation with a freshly prepared mixture of 3.30 -diaminobenzidine tetrahydrochloride and H2O2 in Tris-buffered saline (pH 7.2) for 3 to 4 min. Each analysis was repeated 3 times. To demonstrate their specificity, the primary antibodies were omitted in the analysis, as a negative control. Protein concentrations of LTAH and LTCS were quantified by measuring optical

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density using Kodak 1D Image Analysis software (Eastman Kodak, Rochester, NY). The obtained data were normalized on the basis of glyceraldehydes-3-phosphate dehydrogenase (Sigma, No. G9545) protein expression. 2.4. Enzyme-linked immunosorbent assay procedure Concentrations of LTB4 and LTC4 in the incubation medium were determined using ELISA kits (Cayman Chemical Co, No. 520111 and No. 520211, respectively) according to the manufacturer’s instructions. The standard curve for LTB4 ranged from 1.96 to 1,000 pg/mL, and the effective dose for 50% inhibition (ID50) of assay was 2.5 pg/mL. The intra- and inter-assay coefficients of variation were 4.5% and 7.2%, respectively. The standard curve for LTC4 ranged from 0.98 to 500 pg/mL, and the effective dose for ID50 of the assay was 1.85 pg/mL. The intra-assay and inter-assay coefficients of variation were 4.7% and 6.2%, respectively. 2.5. Statistical analysis Only those results from the incubation of tissues for which the release of PGF2a in response to NONOate was statistically significant were considered. The means  standard error of the mean (SEM) in the CON and E coli groups were calculated from 18 values, with those in the SAL groups based on 16. The data have been presented as means  SEM, with differences accepted to be statistically significant when P < 0.05. Oneway and two-way analysis of variance (day, treatment) followed by the Bonferroni test (InStat Graph Pad, San Diego, CA) were applied to compare the mean values. 3. Results 3.1. The effect of LPS and cytokines on the protein expression of LTAH and LTCS Western blots revealed protein bands that corresponded to the expected molecular weight of LTAH (70 kDa) and LTCS (18 kDa) (Figs. 1 and 2). The bands were absent when the primary antibodies were omitted (data not shown). 3.1.1. LTAH day 0 The expression of the LTAH protein in the endometrium of the CON group was greater (P < 0.01) in response to 10 ng/mL of LPS, 1 and 10 ng/mL of TNF-a, 10 ng/mL of IL-1b, and 1 ng/mL of IL-4 compared with the control value and after using 100 ng/mL of LPS, 1 ng/mL of IL-1b, 10 ng/mL of IL-4, and 1 and 10 ng/mL of IL-10 (Fig. 1A). 3.1.2. LTAH day 8 In the endometrium of the SAL group, the protein amount of LTAH was greater (P < 0.01) after treatment with 10 ng/mL of LPS than in the control sample and in response to 1 ng/mL of TNF-a, as well as 1 and 10 ng/mL of IL-4 (Fig. 1B). Both doses of IL-1b increased (P < 0.001) the endometrial expression of LTAH in the E coli group compared with the untreated tissues and those treated with the remaining factors. In this group, the content of LTAH was also greater (P < 0.01) after applying the lower

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dose of LPS in relation to the control value and after using the higher dose of LPS and the lower dose of IL-10. Compared with the SAL group, the content of LTAH in the E coli group was greater in response to the lower dose of TNF-a (P < 0.001) and both doses of IL-1b (P < 0.001) and IL-4 (1 ng/mL, P < 0.01; 10 ng/mL, P < 0.001). 3.1.3. LTAH day 16 The amount of LTAH protein in the SAL group was greater (P < 0.05) in response to LPS, IL-4, and IL-10 at 10 ng/mL compared with the nontreated tissue, as well as after using the higher dose of TNF-a, both doses of IL-1b, and the lower dose of IL-4 and IL-10 (Fig. 1C). The content of LTAH protein in the inflamed tissue was greater (P < 0.01) in response to the higher dose of LPS and the lower dose of IL-4 in relation to the control value, as well as when compared with the effects of applying the lower dose of LPS, the higher dose of TNF-a, and both doses of IL-1b and IL-10. In comparison with the SAL group, the amount of LTAH protein in the E coli group was greater after treatment with the higher doses of LPS (P < 0.01) and TNF-a (P < 0.05) and lower doses of IL-4 (P < 0.001). 3.1.4. LTCS day 0 In the endometrium of the CON group, the content of the LTCS protein was greater after the application of both doses of TNF-a (P < 0.01) and IL-4 (1 ng/mL, P < 0.01 and 10 ng/mL, P < 0.001) in relation to the control value, as well as after using both doses of LPS, the lower dose of IL-1b, and the higher dose of IL-10 (Fig. 2A). 3.1.5. LTCS day 8 In relation to the control value in the SAL group, our study revealed a greater (P < 0.001) amount of the LTCS protein after the application of both doses of LPS, IL-4, and IL-10 (Fig. 2B). In the inflamed tissue, the amount of LTCS protein was greater (P < 0.01) in response to both doses of LPS, TNF-a, and IL-4 compared with the control value and after the application of both doses of IL-1b. In relation to the SAL group, the amount of LTCS protein in the E coli group decreased along with higher doses of LPS and IL-1b (P < 0.05), both doses of IL-4 (P < 0.05) and a lower dose of IL-10 (P < 0.01). 3.1.6. LTCS day 16 The content of the LTCS protein in the endometrium of the SAL group was greater (P < 0.01) in response to the lower doses of LPS and IL-4, as well as to both doses of IL10, compared with the control value and values obtained after using the higher dose of LPS and both doses of TNF-a (Fig. 2C). The amount of LTCS protein in the E coli group was greater (P < 0.01) in response to all of the analyzed factors compared with the nontreated tissue. In relation to the

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results obtained for the SAL group, the LTCS content in the inflamed endometrium was greater when applying the higher dose of LPS (P < 0.05) and both doses of TNF-a (P < 0.001), IL-1b, and IL-4 (1 ng/mL, P < 0.01 and 10 ng/mL, P < 0.001). 3.2. The effect of LPS and cytokines on the secretion of LTB4 and LTC4 3.2.1. LTB4 day 0 The contents of LTB4 in the medium after incubation of the CON group endometrium were elevated by both doses of LPS (P < 0.01) and by the lower dose of IL-1b (P < 0.01), IL-4 (P < 0.05), and IL-10 (P < 0.01) compared with the control value (Fig. 3A). 3.2.2. LTB4 day 8 The analyzed factors did not significantly affect the secretion of LTB4 from the endometrium of the SAL group (Fig. 3B). The lower dose of TNF-a and the higher dose of IL1b and IL-4 stimulated (P < 0.05) the release of LTB4 from the inflamed tissue in relation to both the control value and after applying the lower dose of LPS. When compared with the SAL group, none of the applied factors revealed a significant effect on the contents of LTB4 in the medium after the incubation of the inflamed endometrium. 3.2.3. LTB4 day 16 No significant differences were noted in the release of LTB4 from the endometrium of the SAL group in response to all of the analyzed factors (Fig. 3C). The higher doses of LPS (P < 0.01) and IL-4 (P < 0.05) increased the secretion of LTB4 from the endometrium of the E coli group in relation to the nontreated tissue and after using the lower doses of LPS and IL-4, both doses of TNF-a and IL-1b, and the higher dose of IL-10. The release of LTB4 from the inflamed endometrium was found to be lower after treatment with TNF-a (1 ng/mL, P < 0.05 and 10 ng/mL, P < 0.01), IL-1b (1 ng/mL, P < 0.01) and IL-10 (10 ng/mL, P < 0.05) than in the SAL group. 3.2.4. LTC4 day 0 The lower dose of LPS stimulated (P < 0.001) LTC4 secretion from the endometrium of the CON group compared with the untreated tissues and those treated with the remaining factors (Fig. 4A). In relation to the control value, the secretion of LTC4 was elevated (P < 0.01) by the higher dose of LPS and by both doses of TNF-a, IL-4, and IL-10. 3.2.5. LTC4 day 8 The medium amounts of LTC4 after the incubation of endometrial tissues from the SAL group with the higher

Fig. 1. The effect of lipopolysaccharide (LPS), tumor necrosis factor a (TNF-a), interleukin (IL) 1b, IL-4, and IL-10 on leukotriene A4 hydrolase (LTAH) protein expression in endometrial explants collected on day 0 from the control gilts (gray bars) and on days 8 and 16 from the saline-treated gilts (white bars) and Escherichia coli-treated gilts(black bars). Values from the densitometric analysis of blots were normalized to GAPDH. Upper panels show representative blots. Data are expressed as means  SEM obtained from six experiments (gilts, in each group). Different letters (A, B, and C) indicate significant differences (P < 0.05–0.001) within the CON, SAL, and E coli groups between the individual treatments. a P < 0.05, b P < 0.01, and c P < 0.001dindicate differences between the SAL and E coli groups subjected to the same treatment. CV, control value (vehicle-treated tissue); CON, control group; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; SAL, saline-treated group; SEM, standard error of the mean.

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(ng/mL) Fig. 3. The effect of lipopolysaccharide (LPS), tumor necrosis factor a (TNF-a), and interleukin (IL) 1b, IL-4, and IL-10 on leukotriene B4 (LTB4) secretion from endometrial explants collected on day 0 from the control gilts (gray bars) and on days 8 and 16 from the saline-treated gilts (white bars) and Escherichia colitreated gilts (black bars). Data are expressed as means  SEM obtained from six experiments (gilts, in each group). Different letters (A and B) indicate significant differences (P < 0.05 and P < 0.01) within the CON, SAL, and E coli groups between the individual treatments. a P < 0.05 and b P < 0.01dindicate differences between the SAL and E coli groups subjected to the same treatment. CV, control value (vehicle-treated tissue); CON, control group; SAL, saline-treated group; SEM, standard error of the mean.

doses of LPS (P < 0.01), TNF-a (P < 0.05), and IL-4 (P < 0.01) and the lower dose of IL-10 (P < 0.01) increased compared with the results from the control medium and those after the application of LPS in the lower dose and IL-1b and IL-10 in the higher doses (Fig. 4B). The release of LTC4 from the inflamed endometrium was higher (P < 0.01) in response to the lower dose of TNF-a and IL-10,

and the higher dose of IL-4, compared with the control value and the effect of other substances, except for the higher dose of TNF-a and lower dose of IL-1b. Larger amounts of LTC4, in relation to the SAL group, were estimated after the incubation of the inflamed endometrium with TNF-a (P < 0.01) and IL-10 (P < 0.05) in their lower doses.

Fig. 2. The effect of lipopolysaccharide (LPS), tumor necrosis factor a (TNF-a), and interleukin (IL) 1b, IL-4, and IL-10 on leukotriene C4 synthase (LTCS) protein expression in endometrial explants collected on day 0 from the control gilts (gray bars) and on days 8 and 16 from the saline-treated gilts (white bars) and Escherichia coli-treated gilts (black bars). Values from densitometric analysis of blots were normalized to GAPDH. Upper panels show representative blots. Data are expressed as means  SEM obtained from six experiments (gilts, in each group). Different letters (A and B) indicate significant differences (P < 0.01 and P < 0.001) within the CON, SAL, and E coli groups between the individual treatments. a P < 0.05, b P < 0.01, and c P < 0.001dindicate differences between the SAL and E coli groups subjected to the same treatment. CV, control value (vehicle-treated tissue); CON, control group; GAPDH, glyceraldehydes-3-phosphate dehydrogenaseSAL, saline-treated group; SEM, standard error of the mean.

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(ng/mL)

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Fig. 4. The effect of lipopolysaccharide (LPS), tumor necrosis factor a (TNF-a), and interleukin (IL) 1b, IL-4, and IL-10 on leukotriene C4 (LTC4) secretion from endometrial explants collected on day 0 from the control gilts (gray bars) and on days 8 and 16 from the saline-treated gilts (white bars) and Escherichia colitreated gilts (black bars). Data are expressed as means  SEM obtained from six experiments (gilts, in each group). Different letters (A and B) indicate significant differences (P < 0.05 and P < 0.01) within the CON, SAL, and E coli groups between the individual treatments. a P < 0.05, b P < 0.01ddifferences between the SAL and E coli groups subjected to the same treatment. CV, control value (vehicle-treated tissue); CON, control group; SAL, saline-treated group; SEM, standard error of the mean.

3.2.6. LTC4 day 16 In the SAL group, the contents of LTC4 in the control medium and after treatment with all of the analyzed factors were similar (Fig. 4C). The release of LTC4 from the inflamed endometrium was greater (P < 0.01) after the application of the higher dose of IL-4 and both doses of IL-10 compared with the control value and after using either dose of IL-1b and the lower dose of IL-4. The inflamed tissue secreted more LTC4 in response to the higher dose of IL-4 (P < 0.01) and IL-10 (P < 0.05) than in the case of the SAL group. 4. Discussion In the present study, we demonstrate that LPS, TNF-a, IL-1b, IL-4, and IL-10 have a significant influence on the

protein expression of LTAH and LTCS in, and the secretion of LTB4 and LTC4 from, the endometrium of an inflamed porcine uterus. The results of macroscopic and histopathologic investigations of uteri used in the previously presented research had been reported in a previous work [6]. Briefly, although acute endometritis was diagnosed in uteri collected on both days 8 and 16 after E coli injections, a more intense inflammatory process occurred more frequently on day 8 than on day 16 of the study. The production of LTs was found to differ between the SAL and E coli-treated groups. On day 8, the amounts of LTAH were higher in the TNF-a–stimulated, IL-1b–stimulated, and IL-4–stimulated inflamed endometrial samples than in those from the saline-injected organ. Contrarily, the protein contents of LTCS were decreased by LPS, IL-1b,

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IL-4, and IL-10 in the E coli-treated endometrium. The expression of both studied enzymes was augmented in response to LPS, TNF-a, and IL-4 in the inflamed endometrium from day 16. On this day, a similar situation was also observed in the case of IL-1b–stimulated LTCS protein expression. These findings are consistent with earlier studies revealing that IL-4 and IL-13 increased the amount of LTAH protein in human neutrophils [12] and that LTCS expression was enhanced by LPS in rat hepatocytes [31] and by IL-4 in human cord blood-derived mast cells [13]. However, data illustrating that IL-4 and IL-13 are able to reduce the amount of LTAH protein in human monocytes during glomerular disease also exists [32]. The present study shows that on day 8 of the study, none of the inflammatory mediators significantly affected LTB4 secretion from the inflamed endometrium compared with the SAL group, whereas LTC4 secretion was increased by TNF-a and IL-10. On day 16, the release of LTB4 from the inflamed tissue was determined to be lower after the application of TNF-a, IL-1b, and IL-10, whereas LTC4 secretion was enhanced by IL-4 and IL-10, in relation to the SAL group. In earlier works, IL-4 and IL-13 have been reported to inhibit the release of LTB4 from human peripheral monocytes [17], whereas activation of eosinophils by IL-1a increased LTC4 secretion from human umbilical vein endothelial cells [33]. Other studies revealed that LTB4 in human neutrophils increases in response to LPS [14] and by IL-1b in human keratinocytes [34]. The influence of inflammatory mediators on the endometrial protein expression of LTAH and LTCS, and on the amounts of LTB4 and LTC4 in the culture medium, was affected by the various treatments, compared with the control values. In the CON group, the expression of both studied enzymes was mainly stimulated by TNF-a and IL-4, whereas the secretion of LTB4 and LTC4 was increased by LPS, IL-4, and IL-10. Moreover, these factors increased the protein expression of LTAH and LTCS (days 8 and 16), and the secretion of LTC4 (day 8), in the SAL group most significantly. None of the applied factors markedly affected the contents of LTB4 in the medium in either of the analyzed periods, nor did they affect the content of LTC4 on day 16. On the other hand, the content of LTAH in and secretion of LTB4 from the inflamed tissues on days 8 and/ or 16 were enhanced predominantly by LPS, IL-1b, and IL-4. In addition to IL-4, LTCS expression in and LTC4 release from the endometrium with inflammation were also stimulated by TNF-a and IL-10. Similarly, LPS increased the release of LTC4 from LPS-induced human amnion cells [35]. Moreover, in bovine mammary gland epithelial cells, the production of LTB4 and LTC4 was stimulated by TNF-a, whereas LPS and IL-1a enhanced the synthesis of LTC4 [36]. The present study also shows that lower and/or higher doses of inflammatory factors affected the endometrial amounts of LTAH and LTCS and the secretion of LTB4 and LTC4 in the particulars groups. However, the levels of studied variables did not rise along with the increasing doses of all applied mediators. In turn, the lower dose of LPS led to the greater increase of the endometrial LTC4 secretion in the CON group than the higher dose. A stimulatory effect of both doses of the analyzed factors was determined more often in the inflamed uteri (on both days) than in the SAL group. A

9

similar situation was also observed in relation to the effects of the lower dose of inflammatory factors on day 8. Additionally, the inflamed uteri responded more frequently to lower or higher doses of factors on day 8 than on day 16. The variation in LT production in uteri from the individual experimental groups in response to both proinflammatory (TNF-a and IL-1b) and anti-inflammatory (IL-4 and IL-10) cytokines may arise from the differential expression of the receptors of these factors. Thus, the greater ability of pro- and anti-inflammatory mediators (their lower and/or higher doses) to stimulate LTB4 and LTC4 synthesis/secretion in the inflamed organs may be associated with the higher sensitivity of such uterine cells to inflammatory mediators. Data reporting upregulation in the protein expression of IL-1 type I and IL-10 receptors in the endometrial tissues of women with endometriosis [37] and adenomyosis [27], respectively, are available. There is also the possibility that cytokines in the inflamed endometrium exerted their effect via multiple signaling pathways involving two or more distinct receptors and numerous intracellular mediators/pathways [38,39]. Immunofluorescence analysis of the inflamed uteri used in the present study indicated that arterial endothelial cells in the endometrium and luminal epithelial cells displaying the overexpression of LTAH and LTCS are the main sources of increased LT synthesis in pathologically changed organs [23]. These cells are likely to be more sensitive to the influence of cytokines, the amounts of which are markedly elevated in the inflamed uterus [18– 22]. What is more, LT production in the inflamed uteri in response to inflammatory factors (the present study) might have occurred in immune cells, mainly neutrophils which are abundant in these organs [6]. The fact that receptors for LPS and cytokines are present in immune cells supports this assumption [40,41]. It should be emphasized that in the present research, IL-1b increased LTAH protein expression in the inflamed endometrium on day 8 more significantly than any of the other applied inflammatory mediators, which may have resulted from the enhanced population of receptors for this cytokine in the pathologically changed tissue. The stronger effect of IL-1b on the secretion of LTB4 in relation to IL-4 and IL-13 had been determined in earlier studies in the monocytes of people with glomerular disease [32]. On the other hand, the release of LTB4 and LTC4 from bovine mammary gland epithelial cells increased more significantly in response to TNF-a than IL-1a and NONOate [36]. Our research revealed that, in the CON, SAL-treated and E coli-treated groups, alterations in the expression of the protein levels of enzymes participating in LT synthesis did not always coincide with the concentrations of these compounds (LTs) found in the medium in response to inflammatory mediators. However, increases in the protein expression of enzymes were more often accompanied by the elevated secretion of their products in the inflamed uteri. The lack of significant changes in the release of LTB4 and LTC4 from the endometrium by the enhanced tissue contents of LTAH and LTCS revealed in our study may be a consequence of LT accumulation in the endometrium, as previously reported in rat hepatocytes [31]. In addition, the synthesized LTC4 may be further metabolized to other

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cysteinyl-LTs, such as LTD4 and LTE4 [9,11]. In turn, the lack of changes in enzyme contents accompanied by an elevated LTs secretion might have resulted from negative feedback between the level of LT protein/gene expression and their product [42]. Our data show that the production of LTs in response to particular factors changed during the course of the study in the SAL and E coli groups. In the endometrium of the SAL group, the expression of LTAH on day 8 was stimulated only by LPS, whereas on day 16, this effect was also exerted by IL-4 and IL-10. In contrast, the content of LTCS was increased by all three of the previously mentioned factors on both study days. As it had been mentioned earlier, the secretion of LTB4 was not altered in either of the experimental periods in the SAL group, whereas the release of LTC4 changed only on day 8. In comparison with the SAL group, the production profiles of LTs differed greatly on both analyzed days of uterine inflammation. Namely, regardless of the research period (days 8 and 16), the applied inflammatory factors stimulated the release of LTs from the inflamed endometrium. On day 8, a higher production of LTB4 was reported mainly after the use of proinflammatory factors, especially IL-1b, although on day 16, higher secretion of LTC4 was noted not only under the influence of proinflammatory (TNF-a and IL-1b) but also anti-inflammatory (IL-4 and IL-10) factors. The greater ability of pro- or anti-inflammatory factors to stimulate LTB4 or LTC4 production on the particular days of uterine inflammation may result from the different expression of receptors for inflammatory factors over the course of the pathological state. Moreover, the somewhat different production of LTs in response to inflammatory mediators on the analyzed days of uterine inflammation may be a consequence of the fact that the hormonal status of gilts on days 8 and 16 of the study (expected days 11 and 19 of the estrous cycle) somewhat varies, which had been reported earlier in gilts with inflamed uteri [5,6,43]. The expression of enzymes synthesizing LTB4 and LTC4 in the equine endometrium had also been reported to change markedly during the estrous cycle [44]. The increase in the synthesis and/or secretion of LTB4 and LTC4 in an inflamed porcine endometrium under the influence of LPS, TNF-a, IL-1b, and IL-4 observed in the present study clearly suggests that these factors may indirectly affect processes regulated by LTs, that is, modulate the inflammatory process. However, as previously mentioned, the higher release of LTB4 on day 8 was observed mainly after the use of proinflammatory factors, whereas on day 16, a greater secretion of LTC4 was noted not only under the influence of proinflammatory (TNF-a and IL1b) but also anti-inflammatory (IL-4 and IL-10) factors. The greater ability of pro- or anti-inflammatory factors to stimulate LTB4 or LTC4 production on the particular days of uterine inflammation may be associated with the function of particular LTs during the inflammatory process. Thus, on day 8, LTB4 contributes to clearing the uterus of E coli, whereas the rise in LTC4 synthesis on day 16 after bacteria treatment suggests its involvement in the course of the later phases of the of inflammation process. LTB4 is involved in the development of the inflammatory response by affecting the migration, activation, and prolongation of inflammatory

1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 5. Conclusions 1150 1151 The presented data provide evidence that proin1152 flammatory (LPS, TNF-a, and IL-1b) and anti-inflammatory 1153 (IL-4 and IL-10) mediators have a stimulatory influence on 1154 the amounts of LTAH and LTCS in, and the secretion of LTB4 1155 and LTC4 from, an inflamed porcine endometrium. How1156 ever, the production of particular LTs is partially dependent 1157 on the type of factors applied and the duration of the in1158 flammatory process (or its intensity). Our research suggests 1159 that by altering LT synthesis/secretion in an inflamed 1160 uterus, the previously mentioned pro- and anti1161 inflammatory factors may indirectly affect the processes 1162 regulated by LTs. Thus, further studies should be carried out 1163 to elucidate the mechanism of LT production in response to 1164 inflammatory mediators in pathologically changed uterine 1165 tissues. 1166 1167 Acknowledgments 1168 1169 This work was supported by a grant from the Polish 1170 Ministry of Scientific Research and Higher Education 1171 (NN308128339). The study will be a part of a PhD thesis by 1172 J. Czarzasta who was supported by the European Union 1173 within the European Social Fund. Q 8 1174 1175 1176 References 1177 [1] Tummaruk P, Kesdangsakonwut S, Prapasarakul N, Kaeoket K. 1178 Endometritis in gilts: reproductive data, bacterial culture, histopa1179 thology, and infiltration of immune cells in the endometrium. Comp 1180 Clin Pathol 2010;19:575–84. 1181 [2] De Winter PJJ, Verdonck M, De Kruif A, Devriese LA, Haesebrouck F. Bacterial endometritis and vaginal discharge in the sow: prevalence 1182 of different bacterial species and experimental reproduction of the 1183 syndrome. Anim Reprod Sci 1995;37:325–35. 1184 [3] Roberson J, Moll D, Saunder G. Chronic Staphylococcus aureus endometritis in virgin gilt. Vet Rec 2007;161:821–2. 1185 [4] Mateus L, Lopes da Costa L, Diniz P, Ziecik AJ. Relationship between 1186 endotoxin and prostaglandin (PGE2 and PGFM) concentrations and 1187 ovarian function in dairy cows with puerperal endometritis. Anim Reprod Sci 2003;76:143–54. 1188 [5] Jana B, Kucharski J, Dzienis A, Deptu1a K. Changes in prostaglandin 1189 production and ovarian function in gilts during endometritis 1190 induced by Escherichia coli infection. Anim Reprod Sci 2007;97: 137–50. 1191 [6] Jana B, Jaroszewski J, Czarzasta J, W1odarczyk M, Markiewicz W. 1192 Synthesis of prostacyclin and its effect on the contractile ac1193 tivity of the inflamed porcine uterus. Theriogenology 2013;79: 470–85. 1194 [7] Peter AT, Bosu WTK, Luker CW. Plasma endotoxin and concentra1195 tions of stable metabolites of prostacyclin, thromboxane A2, and 1196 prostaglandin E2 in postpartum dairy cows. Prostaglandins 1987; 1197 34:15–28. cell survival [45]. In contrast, LTC4, besides participation in the development of inflammation, is also involved in the maintenance and regeneration of damaged tissues [46]. What is more, the increased levels of LTB4 and LTC4, which are present in an inflamed uterus in response to both proand anti-inflammatory mediators may stimulate 11bhydroxysteroid dehydrogenase, which produces cortisol the anti-inflammatory hormone presented earlier [47]. Therefore, inflammatory mediators may increase the uterine production of glucocorticoid not only directly [48,49] but also by the 5-LO pathway.

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[8] Jana B, Andronowska A, Kucharski J. Nitric oxide mediates an inflammatory effect of Escherichia coli in the porcine uterus. Polish J Vet Sci 2000;4:207–12. [9] Peters-Golden M, Henderson WR. Leukotrienesdmechanisms of disease. N Engl J Med 2007;357:1841–54. [10] Rinaldo-Matthis A, Haeggström JZ. Structures and mechanisms of enzymes in the leukotriene cascade. Biochimie 2010;92:676–81. [11] Dighe NS, Pattan SR, Merekar AN, Dighe SB, Chavan PA, Musmade DS, Gaware VM. Leukotrienes and its biological activities. A Rev J Chem Pharm Res 2010;2:338–48. [12] Zaitsu M, Hamasaki Y, Matsuo M, Kukita A, Tsuji K, Miyazaki M, Hayasaki R, Muro E, Yamamoto S, Kobayashi I, Ichimaru T, Kohashi O, Miyazaki S. New induction of leukotriene A(4) hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes. Blood 2000;96:601–9. [13] Hsieh FH, Lam BK, Penrose JF, Austen KF, Boyce JA. T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4. J Exp Med 2001;193:123–33. [14] Sinclair R, Eriksson AS, Gretzer C, Cassuto J, Thomsen P. Inhibitory effects of amide local anaesthetics on stimulus-induced human leukocyte metabolic activation, LTB4 release and IL-1 secretion in vitro. Acta Anaesthesiol Scand 1993;37:159–65. [15] Yudina Y, Parhamifar L, Bengtsson AM, Juhas M, Sjölander A. Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells. Prostaglandins Leukot Essent Fatty Acids 2008;79:223–31. [16] Edwin SS, LaMarche SL, Thai D, Branch DW, Mitchell MD. 5Hydroxyeicosatetraenoic acid biosynthesis by gestational tissues: effects of inflammatory cytokines. Am J Obstet Gynecol 1993;169: 1467–71. [17] Nassar GM, Montero A, Fukunaga M, Badr KF. Contrasting effects of proinflammatory and T-helper lymphocyte subset-2 cytokines on the 5-lipoxygenase pathway in monocytes. Kidney Int 1997;51: 1520–8.  ska-Krakowska M, Wolin  ska-Witort E. The [18] Jana B, Kucharski J, Jedlin effect of intrauterine infusion of inflammation-provoking factors on proinflammatory cytokines and hormones in rat peripheral blood. Polish J Vet Sci 2005;4:275–82. [19] Herath S, Fischer DP, Werling D, Williams EJ, Lilly ST, Dobson H, Bryant CE, Sheldon IM. Expression and function of toll-like receptor 4 in the endometrial cells of the uterus. Endocrinology 2006;147: 562–70. [20] Gabler C, Drillich M, Fischer C, Holder C, Heuwieser W, Einspanier R. Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis. Theriogenology 2009; 71:993–1004. [21] Shao CY, Wang H, Meng X, Zhu JQ, Wu YQ, Li JJ. Characterization of the innate immune response in goats after intrauterine infusion of E. coli using histopathological, cytologic and molecular analyses. Theriogenology 2012;78:593–604. [22] Woodward EM, Christoffersen M, Campos J, Betancourt A, Horohov D, Scoggin KE, Squires EL, Troedsson MH. Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis. Reproduction 2013;145:289–96. [23] Jana B, Czarzasta J, Jaroszewski J. Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli. Reprod Fertil Dev 2013. http://dx.doi.org/10.1071/ RD13191. In Press. [24] Okuda K, Sakumoto R, Okamoto N, Acosta TJ, Abe H, Okada H, Sinowatz F, Skarzynski DJ. Cellular localization of genes and proteins for tumor necrosis factor-a (TNF), TNF receptor types I and II in bovine endometrium. Mol Cell Endocrinol 2010;330:41–8. [25] Seo H, Choi Y, Shim J, Choi Y, Ka H. Regulatory mechanism for expression of IL1b receptors in the uterine endometrium and effects of IL1b on prostaglandin synthetic enzymes during the implantation period in pigs. Biol Reprod 2012;87:1–11. [26] De Moraes-Pinto MI, Vince GS, Flanagan BF, Hart CA, Johnson PM. Localization of IL-4 and IL-4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunology 1997;90: 87–94. [27] Qin X, Zhang H, Wang F, Xue J, Wen Z. Expression and possible role of interleukin-10 receptors in patients with adenomyosis. Eur J Obstet Gynecol Reprod Biol 2012;161:194–8.

11

[28] Roberto da Costa RP, Costa AS, Korzekwa AJ, Platek R, Siemieniuch M, Galvão A, Redmer DA, Silva JR, Skarzynski DJ, Ferreira-Dias G. Actions of a nitric oxide donor on prostaglandin production and angiogenic activity in the equine endometrium. Reprod Fertil Dev 2008;20:674–83.  ska-Krakowska M. The [29] Jana B, Koz1owska A, Andronowska A, Jedlin effect of tumor necrosis factor-a (TNF-a), interleukin (IL)-1b and IL6 on chorioamnion secretion of prostaglandins (PG)F2a and E2 in pigs. Reprod Biol 2008;8:57–68. [30] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248–54. [31] Shimada K, Navarro J, Goeger DE, Mustafa SB, Weigel PH, Weinman SA. Expression and regulation of leukotriene-synthesis enzymes in rat liver cells. Hepatology 1998;28:1275–81. [32] Montero A, Nassar GM, Uda S, Munger KA, Badr KF. Reciprocal regulation of LTA4 hydrolase expression in human monocytes by g-interferon and interleukins 4 and 13: potential relevance to leukotriene regulation in glomerular disease. Exp Nephrol 2000;8: 258–65. [33] Muñoz NM, Hamann KJ, Rabe KF, Sano H, Zhu X, Leff AR. Augmentation of eosinophil degranulation and LTC(4) secretion by integrin-mediated endothelial cell adhesion. Am J Physiol 1999; 277:L802–10. [34] Sjursen W, Brekke OL, Johansen B. Secretory and cytosolic phospholipase A2 regulate the long-term cytokine-induced eicosanoid production in human keratinocytes. Cytokine 2000;12:1189–94. [35] Shon YH, Kim JH, Nam KS. Effect of astragali radix extract on lipopolysaccharide-induced inflammation in human amnion. Biol Pharm Bull 2002;25:77–80. [36] Piotrowska-Tomala KK, Siemieniuch MJ, Szóstek AZ, Korzekwa AJ, Woclawek-Potocka I, Galváo AM, Okuda K, Skarzynski DJ. Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells. Domest Anim Endocrinol 2012;43:278–88. [37] Akoum A, Lawson C, Herrmann-Lavoie C, Maheux R. Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis. Hum Reprod 2007;22: 1464–73. [38] Dinarello CA. The interleukin-1 family: 10 years of discovery. FASEB J 1994;8:1314–25. [39] Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today 1992; 13:151–3. [40] Mashima R, Saeki K, Aki D, Minoda Y, Takaki H, Sanada T, Kobayashi T, Aburatani H, Yamanashi Y, Yoshimura A. FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling. J Biol Chem 2005;280: 41289–97. [41] Lacy P, Stow JL. Cytokine release from innate immune cells: association with diverse membrane trafficking pathways. Blood 2011; 118:9–18. [42] Wathes DC, Hamon M. Localisation of oestradiol, progesterone and oxytocin receptors in the uterus during the oestrous cycle and early pregnancy of the ewe. J Endocrinol 1993;138:479–91. [43] Jana B, Kucharski J, Ziecik AJ. Effect of intrauterine infusion of Escherichia coli on hormonal patterns in gilts during the oestrous cycle. Reprod Nutr Dev 2004;44:37–48. [44] Guzeloglu A, Atli MO, Kurar E, Kayis SA, Handler J, Semacan A, Aslan S. Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy. Theriogenology 2001;80:145–52. [45] Tager AM, Luster AD. BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins Leukot Essent Fatty Acids 2003;69:123–34. [46] Kanaoka Y, Boyce JA. Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses. J Immunol 2004;173:1503–10. [47] Sato K, Chisaka H, Okamura K, Challis JR. Effect of the interaction between lipoxygenase pathway and progesterone on the regulation of hydroxysteroid 11-beta dehydrogenase 2 in cultured human term placental trophoblasts. Biol Reprod 2008;78:514–20. [48] Murphy SP, Tayade C, Ashkar AA, Hatta K, Zhang J, Croy BA. Interferon gamma in successful pregnancies. Biol Reprod 2009;80: 848–59. [49] Monsivais D, Bray JD, Su E, Pavone ME, Dyson MT, Navarro A, Kakinuma T, Bulun SE. Activated glucocorticoid and eicosanoid pathways in endometriosis. Fertil Steril 2012;98:117–25.

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