Elsevier

Theriogenology

Volume 83, Issue 4, 1 March 2015, Pages 596-603
Theriogenology

Research article
The role of toll-like receptors 2 and 4 in the pathogenesis of feline pyometra

https://doi.org/10.1016/j.theriogenology.2014.10.023Get rights and content

Abstract

Pyometra is the most common uterine disease in queens. To protect itself from infection, the female reproductive tract possesses several immune mechanisms that are based on germline-encoded pattern recognition receptors (toll-like receptors [TLRs]). The aim of our study was to examine endometrial immunolocalization of TLR2/4, study the influence of lipopolysaccharide (LPS) and tumor necrosis factor (TNF) α on messenger RNA expression of both receptors in pyometric queens, and compare these patterns between estrous cycling queens and those hormonally treated with medroxyprogesterone acetate (MPA). Thirty-six queens, ranging in age from 7 months to 11 years, were allocated into seven groups (anestrus, estrus, mid-diestrus and late diestrus, short-term and long-term hormonally treated queens, and pyometric queens). At the messenger RNA level, the real-time polymerase chain reaction was applied, whereas at the TLR2/4 protein level, the expression was tested by immunohistochemistry. In queens at estrus, gene expression of TLR2 was upregulated after stimulation of endometrial explants by TNF (P < 0.001) and by TNF together with the LPS (P < 0.01). Moreover, gene expression of TLR2 was significantly upregulated after stimulation by TNF (P < 0.001) and LPS (P < 0.01) explants derived from queens that had been long-term hormonally treated with MPA. Endometrial gene expression of TLR4 was significantly upregulated after incubation of explants with TNF (P < 0.001) in queens at estrus and with LPS (P < 0.05) in queens short-term hormonally treated with MPA. Immunolocalization reported that TLR2/4 receptors are mainly localized in the surface and glandular epithelia. These data show that short-term and especially long-term administration of progesterone derivatives impairs TLRs in the endometrial epithelium, presumably enabling pathogens to break through this first natural barrier and thereby increase the risk of pyometra development.

Introduction

Hormonal contraception designed to control reproductive activity by preventing heat may frequently cause pyometra. Changes in the uterine microenvironment and decreased contractility assist bacterial invasion [1]. The uterine lumen is usually free from pathogenic bacteria; however, the lower parts of the genitourinary tract are readily colonized by opportunistic bacteria. Closure of the uterine cervix normally protects the uterine lumen against bacteria invading from the vagina. However, in females in heat and in periparturient animals, the uterine cervix is open, facilitating bacterial invasion into the endometrium. During pyometra, the most common bacterium isolated from the feline uterus is Escherichia coli, followed by Streptococcus spp., Klebsiella spp., Staphylococcus aureus, Pasteurella spp., Proteus spp., and Pseudomonas spp. [2]. To protect the female reproductive tract against infection, several innate immune mechanisms have developed. The first line of protection against pathogens is a mucous membrane [3]. The mucosal epithelial cells are in constant contact with microbial flora within the female reproductive tract and recognize various components of bacterial, fungal, and viral pathogens commonly known as pathogen-associated molecular patterns through pattern recognition receptors, which include the toll-like receptors (TLRs) family [4]. Nine types of TLRs (TLR 1–9) have been described in feline tissues [5], [6]. Bacterial cell wall components binding to the respective TLRs initiate an early nonspecific immune response. Toll-like receptor 2 recognizes lipoteichoic acid from gram-positive bacteria and lipoproteins/lipopeptides from gram-negative and gram-positive bacteria [7], whereas TLR4 connects with lipopolysaccharide (LPS) from gram-negative bacteria and heat shock proteins [8]. Activation of TLR4 by LPS causes increased secretion of cytokines such as tumor necrosis factor (TNF) α and chemokines [9]. It has been proved that TNF can be synthesized not only by immune-competent cells such as macrophages and monocytes but also by epithelial cells in the feline endometrium under the influence of LPS [10].

We hypothesized that in cats receiving progestagens as a hormonal contraceptive, the resulting alterations in TLR2 and TLR4 expression are involved in the pathogenesis of endometritis and accompany the pyometra. Therefore, the aim of our study was to examine endometrial immunolocalization of TLR2/4 and study gene expression of both receptors after stimulation with TNF and LPS in pyometric queens or queens hormonally treated with medroxyprogesterone acetate (MPA) and in reproductively healthy queens throughout the estrous cycle.

Section snippets

Animals

All procedures were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (No. 60/2010/DTN).

Thirty-six queens aged between 7 months and 11 years (average, 2.67 years) were ovariohysterectomized. To assign each animal to the appropriate group (anestrus, estrus, mid-diestrus and late diestrus, short-term and long-term hormonally treated queens, and pyometric queens), information was obtained from macroscopic observations of the ovaries as previously described [11], from

TLR2 and TLR4 gene expression

Figure 1 shows n-fold changes in mRNA level expression profiles of TLR2 and TLR4 in epithelial and stromal cells after 2, 4, or 12 hours of stimulation with TNF and LPS. Incubating epithelial cells with LPS increased expression of TLR2 after 12 hours (P < 0.001) and expression of TLR4 after 4 and 12 hours (P < 0.001 and P < 0.01, respectively) compared with the control. Expression of TLR2 was upregulated when stromal cells were stimulated by TNF and peaked at 2 and 12 hours (P < 0.01 and

Discussion

The uterus is a dynamic organ under the control of estradiol and progesterone during the estrous cycle. Commonly observed in veterinary practice are endometrial inflammations in the luteal phase in dogs and cats [14], [15] or during the postparturient period in farm animals [9], [15]. Moreover, pyometritis is usually not observed at estrus [9], [14], [16], clearly indicating that the incidence of uterine inflammation is connected with priming of the uterus with progesterone. The effect of

Acknowledgments

The present study was done with support of the Polish Ministry of Scientific Research and High Education (N308 560 740). The authors thank the DOGMED Veterinary Clinic and Department of Animal Reproduction with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland, for providing materials used in this study. Authors are grateful to Dr. Barry Bavister for careful editing of the article. E. Jursza was supported by the European Union within the European Social

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