Inflammation changes the expression of leukotriene receptors in porcine uteri

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Abstract

Leukotrienes (LTs) are important as pro-inflammatory mediators to the innate immune response, inflammation, and allergy. Although it is known that inflammation of the uterus in gilts leads to an increase in LTB4 and LTC4 synthesis, researchers lack knowledge regarding the expression of LT receptors in the inflamed uterus. The aim of this experiment was to determine the levels of cysteinyl LT receptors type 1 (CysLT(1)R) and type 2 (CysLT(2)R), as well as of LTB4 receptor type 1 (LTB4R1) mRNA and protein expression, and the cellular localization of these receptors in the inflamed porcine uterus. On Day 3 of the estrous cycle (Day 0 of the study), 50 ml of either saline or Escherichia coli suspension (109 colony-forming units/ml) was injected into each uterine horn. Injections of bacteria increased CysLT(1)R mRNA levels in the endometrium on Days 8 and 16 of the study and in the myometrium on Day 16, as well as the CysLT(1)R protein level in the endometrium on Day 8. In the inflamed uteri, CysLT(2)R mRNA levels in the myometrium were increased on Days 8 and 16; however, the protein levels of this receptor decreased in the endometrium (Day 16) and myometrium (Day 8). After bacterial treatment, LTB4R1 mRNA and protein levels in the endometrium on Day 8, and LTB4R1 mRNA levels in the myometrium on Days 8 and 16 were found to have increased. Data obtained show that inflammation of porcine uteri changes the expression of LT receptors, suggesting their importance in the course/consequences of uterine inflammation.

Introduction

Inflammation of the uterus (metritis/endometritis) is one of the most common reproductive disorders in female domestic animals. Consequences of this disorder are varied and can range from no effect on reproductive performance to perpetual sterility. There are a lot of reasons behind culling gilts with endometritis, such as abnormal vaginal discharge, abortion, no pregnancy and anestrus (Tummaruk et al., 2010). The following bacteria were isolated most often from the uteri of sows, both in the absence or presence of endometritis: Escherichia coli (E. coli), Staphylococcus spp., Streptococcus spp., Enterococcus sp., Corynebacterium sp. (De Winter et al., 1995, Roberson et al., 2007, Tummaruk et al., 2010). Endometritis leads to considerable changes in the synthesis and release of inflammatory mediators, i.e., prostaglandin (PG) F2α, PGE2 (Mateus et al., 2003, Jana et al., 2007), PGI2 (Jana et al., 2013a), thromboxane A2 (Peter et al., 1987), and nitric oxide (NO) (Jana et al., 2000), as well as pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 (Jana et al., 2005, Herath et al., 2006, Gabler et al., 2009, Shao et al., 2012).

Leukotrienes (LTs) belong to a family of pro-inflammatory lipid mediators derived from arachidonic acid (AA), which is released from the cell membrane under the influence of phospholipase A2. The synthesis of LTs is initiated by a two-step process of converting AA into the unstable epoxide intermediate, LTA4, which is catalyzed by 5-lipoxygenase (5-LO) in concert with 5-LO-activating protein (FLAP). LTA4 is further metabolized by LTA4 hydrolase (LTAH) to LTB4 or conjugated to tripeptide glutathione by LTC4 synthase (LTCS) to form LTC4. LTC4 and its metabolites, LTD4 and LTE4, are collectively termed the Cysteinyl-LTs (Cys-LTs) (Peters-Golden and Henderson, 2007, Rinaldo-Matthis and Haeggström, 2010, Dighe et al., 2010). The biological effects of LTs are mediated by specific 7-transmembrane G protein-coupled receptors: LTB4R1/LTB4R2 for LTB4 and CysLT(1)R/CysLT(2)R for Cys-LTs (Yokomizo et al., 2000, Capra, 2004). The distribution of LT receptors in selected cells/organs and the effects of LTs mediated by these receptors are given in Table 1.

In the available literature, there is a paucity of data concerning the synthesis, secretion, and role of LTs in the uterus. It was reported that, under physiological conditions, LTC4-binding sites are expressed in human (Chegini and Rao, 1988a) and bovine (Chegini and Rao, 1988b) uterine tissues and that Cys-LTs increase the contractility of the porcine uterus (Ledwozyw and Kodziołka, 1989). When analyzing uteri under inflammatory conditions, earlier studies conducted by the authors of the present article are the only available research to reveal that intrauterine E. coli injections in gilts elevated the levels of LTB4 and LTC4 in peripheral blood (B. Jana, unpublished data) as well as the contents of these LTs and the expression of 5-LO, LTAH, and LTCS in the uterine tissues (Jana et al., 2013b). Moreover, Czarzasta observed that LTC4 increased the contraction activity of the inflamed porcine uterus (unpublished data). Based on the above-mentioned data, we hypothesize that the developing uterine inflammation can also lead to changes in the expression of LT receptors, which may play an important role in the course and/or outcomes of this pathological state. Therefore, the effect of the intrauterine injection of E. coli suspension in gilts on: (1) the levels of CysLT(1)R, CysLT(2)R, and LTB4R1 mRNA and protein expression, and (2) the cellular localization of these receptors, was subjected to examination.

Section snippets

Animals and experimental procedures

All animals were housed and treated in accordance with procedures that had the consent of the Local Ethics Committee (conforming to 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 7–8 months and weighing between 107 and 122 kg. Behavioral estrus was determined with the aid of a tester boar. The animals originated from a herd that had been characterized by an absence of disturbances in

Polymerase chain reaction

The levels of CysLT(1)R mRNA expression in the endometrium of the saline-treated uteri did not change statistically on Days 8 and 16 compared with the control organs (Day 0 of the study; Fig. 1A). In the endometrium of the inflamed uteri the values of CysLT(1)R mRNA were higher (P < 0.001) than in the control uteri on both Days 8 and 16. The levels of CysLT(1)R mRNA expression in the endometrium of the E. coli-injected uteri were augmented (P < 0.001) on Days 8 and 16 compared with the

Discussion

The results of the present study show the changes in the expression patterns of CysLT(1)R, CysLT(2)R, and LTB4R1 in the inflamed uteri of gilts. Macroscopic and histopathological examination of the uteri used in the present study had been described earlier. Briefly, macro- and microscopic evaluation did not reveal any inflammatory changes in the saline-treated (Days 8 and 16 of the study) and the control (Day 0 of the study) uteri. In uteri collected on Days 8 and 16 after E. coli injections,

Acknowledgments

This work was supported by a grant from the Polish Ministry of Scientific Research and Higher Education (NN308128339). This study will be a part of a PhD thesis by J. Czarzasta. J. Czarzasta was supported by the European Union within the European Social Fund (Dr INNO 3).

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