Page 1 of 37 Reproduction Advance Publication first posted on 2 June 2014 as Manuscript REP-14-0163

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Innate immunity and inflammation of the bovine female reproductive tract in health and disease Short title: Immunity and inflammation in reproduction 1

I. Martin Sheldon, 1 James G. Cronin, 1 Gareth D. Healey, 2 Christoph Gabler, 3 Wolfgang Heuwieser, 4 Dominik Streyl, 5 John J Bromfield, 6 Akio Miyamoto, 7 Chrys Fergani, 7 Hilary Dobson 1

College of Medicine, Swansea University, Swansea, SA2 8PP, UK Institute of Veterinary Biochemistry, 3 Clinic of Animal Reproduction, Freie Universitaet Berlin, Berlin, Germany 4 Clinic for Ruminants with Ambulatory and Herd Health Services, Centre for Clinical Veterinary Medicine, Ludwig Maximilian University of Munich, Oberschleißheim, Germany 5 Department of Animal Sciences, University of Florida, Gainesville FL 32608, USA 6 Graduate School for Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan 7 School of Veterinary Science, University of Liverpool, Leahurst, Neston, CH64 7TE, UK 2

[email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] ; [email protected]; [email protected]; [email protected]

Disclosure summary: The authors have nothing to disclose Acknowledgements: This work was initiated following the International Conference on Animal Reproduction 2012 and we thank all the members of our community for useful discussions, including Heinrich Bollwein, Gaetano Donofrio, Marc Drillich, Jocelyn Dubuc, Alex Evans, Robert Gilbert, Doug Hammon, Peter Hansen, Bernd Helms, Giovanna Lazzari, Martina Hoedemaker, Stephen LeBlanc, Matt Lucy, Gaetano Mari, Scott McDougall, Michael McGowan, Deborah Nash, David Noakes, Lilian Oliveira, Joy Pate, Olivier Sandra, Jose Santos, Hans-Joachim Schuberth, David Smith, Tom Spencer, William Thatcher, Erin Williams, Milo Wiltbank, Peter Vos and Holm Zerbe. We apologise to those whose work we were unable to cite for limitation of space. The author is supported in part by the U.K. Biotechnology and Biological Sciences Research Council (www.bbsrc.ac.uk grant numbers BB/I017240/1 and BB/K006592/1).

Key words: Cow, inflammation, innate immunity, hypothalamus, uterus, ovary, oocyte, infection. Précis: Inflammatory mediators play key roles in reproductive physiology and the maintenance of tissue homeostasis, but innate immunity and inflammation are also central to the defence of the reproductive tract against infection. Address correspondence to: Professor Iain Martin Sheldon, Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, UK. E-mail: [email protected]

1 Copyright © 2014 by the Society for Reproduction and Fertility.

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Abstract

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Mammalian reproductive physiology and the development of viviparity co-evolved with

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inflammation and immunity over millennia. Many inflammatory mediators contribute to

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paracrine and endocrine signalling, and the maintenance of tissue homeostasis in the female

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reproductive tract. However, inflammation is also a feature of microbial infections of the

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reproductive tract. Bacteria and viruses commonly cause endometritis, perturb ovarian follicle

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development, and suppress the endocrine activity of the hypothalamus and pituitary in cattle.

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Innate immunity is an evolutionary ancient system that orchestrates host cell inflammatory

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response aimed at eliminating pathogens and repairing damaged tissue. Pattern recognition

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receptors on host cells bind pathogen-associated molecular patterns and damage-associated

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molecular patterns, leading to activation of intracellular MAPK and NFκB signalling

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pathways, and the release of inflammatory mediators. Inflammatory mediators typically

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include the interleukin cytokines IL-1β and IL-6, chemokines such as IL-8, interferons, and

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prostaglandins. This review outlines the mechanisms of inflammation and innate immunity in

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the bovine female reproductive tract during health and disease.

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Introduction

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Inflammation is a cellular reaction triggered by noxious stimuli, including infection and tissue

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injury, which evolved as an adaptive response for restoring homeostasis in the cells and tissue

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of simple organisms (Medzhitov 2008). Prior to the appearance of receptors and signalling

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cascades, cytokines evolved as intracellular messengers in invertebrates, where they play a

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role in host defence and repair (Dinarello 2007). The general term “cytokine” includes several

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families of proteins including interleukins, chemokines, interferons, mesenchymal growth

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factors, adipokines and tumour necrosis factors, whilst typical lipid mediators of

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inflammation include prostaglandins and resolvins. Most of these inflammatory mediators are

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soluble factors that are produced by one cell and act on another target cell to change its

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function; some consider these mediators as the “hormones” of the immune and inflammatory

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responses (Dinarello 2007). The systems of inflammation and innate immunity have co-

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evolved with reproductive physiology and the development of viviparity over 340 million

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years. Several inflammatory mediators were co-opted and used for signalling processes during

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evolution of the reproductive system, and have primary roles in physiology rather than

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inflammation. Inflammation is also part of maintaining tissue homeostasis during

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physiological changes in architecture, such as ovulation or parturition. However, innate

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immunity remains vital to protect the female reproductive tract from the adverse effects of

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microbial infections. These infections are particularly important in Bos taurus, where bacteria

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and viruses commonly cause infertility. This review outlines the mechanisms of inflammation

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and innate immunity in the bovine female reproductive tract during health and disease.

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Inflammation

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After infection or tissue injury, inflammatory mediators are released resulting in the cardinal

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signs of inflammation: redness, heat, swelling, pain and loss of function. Inflammatory

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mediators typically include cytokines such as interleukin (IL)-1β, IL-6 and tumour necrosis

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factor (TNFα), chemokines such as IL-8, and prostaglandin E2. These mediators initially

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direct the inflammatory response, by attracting and activating immune cells, particularly

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neutrophils and macrophages to remove microbes and damaged host cells. Later,

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inflammatory mediators such as IL-10 and resolvins foster the repair of tissue, and coordinate

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the timely resolution of inflammation. However, multiple cytokines are involved in the

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inflammatory response, many have pleiotropic effects, and they often duplicate the function

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of each other.

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Innate immunity

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Innate immunity encompasses several facets of non-specific protection against microbial

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infection and tissue damage in the female reproductive tract. The obvious anatomical barriers

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comprising the vulva, vagina and cervix counter ascending microbial infections reaching the

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uterus. Other physical barriers in the genital tract include the stratified squamous epithelium

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of the vagina, the columnar epithelium of the endometrium, the basement membrane of

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ovarian follicles, and the zona pellucida of the oocyte. Antimicrobial peptides and mucosal

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glycoproteins also cover the mucosa to neutralise bacteria and prevent them reaching the

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epithelium. The principal cysteine-rich, cationic, antimicrobial peptides expressed in the

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bovine endometrium include β-defensins, lingual antimicrobial peptide (LAP) and tracheal

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antimicrobial peptide (TAP), and their transcripts are more abundant in the face of microbial

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challenge (Chapwanya, et al. 2013, Davies, et al. 2008). Other surface molecules that may

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help protect the endometrium include the mucins; evidence for their role includes genetic

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deletion of Muc1 in mice, which is associated with chronic inflammation of the lower female

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reproductive tract by opportunistic bacterial infections (DeSouza, et al. 1999). The expression

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of mRNA encoding acute phase proteins in the uterus and ovary is also of interest because

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they may provide further localized protection (Chapwanya, et al. 2013, Fischer, et al. 2010,

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Lecchi, et al. 2012). Although, it is possible that the more usual hepatic production of acute

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phase proteins in response to increased serum concentrations of IL-6 may be more relevant in

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vivo. The Complement system is also present in the female reproductive tract, and this series

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of related proteins opsonise infected cells to attract immunoglobulin and drive the formation

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of the membrane attack complex leading to cytolysis. However, normal cells in the

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reproductive tract are protected against formation of the complement complex by complement

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regulatory proteins including CD46, CD55 and CD59 (Jensen, et al. 1995).

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Whilst physical barriers and secreted proteins are important for passive defence against

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microbes in the female reproductive tract, there are some caveats. First, microbes have

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evolved strategies to avoid many host defences. For example, some bacteria produce enzymes

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that digest mucus to penetrate the protective layer, and many bacteria secrete proteases that

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can disrupt protective peptides and proteins produced by the host (Baxt, et al. 2013). Second,

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the rapid inflammatory responses associated with microbial infections are associated with

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pattern recognition receptors of the innate immune system, rather than passive defence

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systems. Finally, there is also a role for adaptive immunity in the bovine reproductive tract,

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with the presence of plasma cells in the endometrium and accumulation of immunoglobulins

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(Anderson, et al. 1996). However, the mechanisms of adaptive immunity in the female

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reproductive tract are not part of the present review

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Innate immunity is predicated on host cells proteins, called pattern recognition receptors that

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are able to sense pathogens or danger. Innate immunity is evolutionary conserved across

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animal phyla, and the first pattern recognition receptor to be identified was Toll in Drosophila

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melanogaster (Lemaitre, et al. 1996). Toll-like receptors (TLRs) and Nod-like receptors

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(NLRs) are the two most studied families of pattern recognition receptors in mammals, and

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some of their ligands are listed in Table 1. The TLRs and NLRs principally bind pathogen

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associated molecular patterns (PAMPs) found in prokaryotes but not eukaryotes (Beutler

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2009, Takeuchi and Akira 2010). However, danger signals and endogenous damage

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associated molecular patterns (DAMPs), such as high-mobility group box 1 protein

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(HMGB1), ATP, nucleic acids and IL-1α, released from damaged or dead cells, are also

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thought to bind pattern recognition receptors to initiate an inflammatory response (Chen and

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Nunez 2010). The most important TLRs for recognition of bacteria are TLR1, TLR2, and

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TLR6, which form heterodimers to bind bacterial lipopeptides (Fig. 1), and TLR4, which

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binds the cell wall component of Gram-negative bacteria lipopolysaccharide (LPS,

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endotoxin), in complex with CD14 and MD-2 (Takeuchi and Akira 2010). The NLRs such as

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NODs and NLRP3, are intra-cytoplasmic receptors that recognize endogenous molecules as

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well as microbial molecules. Binding of PAMPs to pattern recognition receptors stimulates

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the production of inflammatory mediators, which recruit neutrophils and monocytes to the site

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of infection (Beutler 2009, Takeuchi and Akira 2010). In addition, NLR activation leads to

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formation of the multi-protein inflammasome complex, characteristically leading to cleavage

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of Caspase-1 to an active form and production of mature IL-1β (Schroder and Tschopp 2010).

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The innate immune response to PAMPs and DAMPs is most commonly driven by

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macrophages, neutrophils and dendritic cells. However, a range of cells other than

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“professional” immune cells also expresses pattern recognition receptors. For example,

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endometrial epithelial and stromal cells express the TLR4/CD14/MD-2 receptor complex

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necessary to detect LPS, and respond to LPS by secreting IL-6, IL-8, and prostaglandin E2

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(Cronin, et al. 2012, Herath, et al. 2009a). So, the overall inflammatory response to pathogens

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associated with innate immunity depends on the sum of the actions of multiple cell types

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rather than just specialised immune cells. Furthermore, host cellular responses to pathogens

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are greater than when the same cells encounter commensal microbes. The impact of

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pathogens on the host is enhanced by a multitude of virulence factors including pore-forming

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toxins, bacterial secretion systems, and bacterial proteases (Blander and Sander 2012). In

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addition, unlike commensal organisms, pathogens not only colonize tissues but they also

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invade the cells of the female reproductive tract, often causing cell death (Donofrio, et al.

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2007, Sheldon, et al. 2010). Therefore, the scale of the inflammatory response to microbes is

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dependent on multiple factors including microbial virulence effectors, host tissue factors, and

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on the regulation of intracellular signalling pathways associated with innate immunity.

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The adaptor molecule MyD88 is essential for downstream signalling of all TLRs, with the

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exception of TLR3, although additional adaptor proteins are also involved with some of the

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TLR dimers (Fig. 1) (Moresco, et al. 2011, Takeuchi and Akira 2010). Once activated,

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MyD88 stimulates IL-1R-associated kinases, which in turn activate TNFR-associated factor 6

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(TRAF6), which catalyses a complex consisting of TGFβ-activated kinase 1 (TAK1) and

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members of the TAK1 binding protein (TAB) family. This complex activates two important

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pathways in inflammation, the MAP kinase pathway and the NFκB pathway. The MAP

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kinase cascade (JNK, ERK1/2 and p38) activates the transcription factor AP-1, which along

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with NFκB drive transcription in the nucleus (Fig. 1). Typical genes transcribed by activation

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of these MAPK and NFκB signalling pathways from surface TLRs include cytokines (IL1A,

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IL1B, IL6 and TNFA), chemokines (CXCL8 and CXCL1), and lipid mediators (PLA2, PGHS

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and PGES). On the other hand, internal TLRs activate signalling pathways in response to

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microbial nucleotides that lead to expression of type I interferons (Fig. 1).

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Inflammation and reproductive physiology

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There are extensive reviews about the role of inflammatory mediators and inflammation in

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reproductive physiology (Jabbour, et al. 2009, Richards, et al. 2008, Richards, et al. 2002,

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Turner, et al. 2012). In particular, physiological events within the ovary, including ovulation

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and corpus luteal formation and regression, are inflammatory-like events (Espey 1980).

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Whilst the LH surge is the main trigger for ovulation, inflammatory mediators regulate

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several of the events around the time of ovulation. For example, IL-6 regulates ovarian

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cumulus granulosa cell function, cumulus-oocyte complex expansion, and oocyte

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competence, at least in mice (Liu, et al. 2009). The process of ovulation involves rupture of

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the dominant follicle, which causes tissue damage, and so it is not surprising that innate

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immunity and inflammation have roles in maintaining tissue homeostasis in the ovary

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(Richards, et al. 2008). In the mouse, TLR4 may have a role in normal cumulus-oocyte

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complex expansion and ovulation by binding endogenous ligands such as hyaluronic acid

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(Shimada, et al. 2008). After ovulation the oocyte enters the uterine tube (oviduct), and

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although many neutrophils are present, prostaglandin E2 released by uterine tube epithelial

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cells after LH stimulation suppresses the phagocytosis of sperm by neutrophils, thereby

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supporting sperm survival in the uterine tube (Marey, et al. 2014). As well as responding to

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LH, the uterine tube epithelial cells have innate immune capabilities, expressing TLR2 and

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TLR4, and generating inflammatory responses to LPS (Kowsar, et al. 2013). The LH surge

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also stimulates localised production of IL-8 and angiogenic factors, basic fibroblast growth

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factor and vascular endothelial growth factor A, which are essential for the development of

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the corpus luteum (Berisha, et al. 2006, Jiemtaweeboon, et al. 2011, Schams, et al. 2001).

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Lymphangiogenesis is also important in the bovine corpus luteum, with expression of

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lymphangiogenic factors particularly in the corpus luteum of mid-cycle as well as early

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pregnancy (Nitta, et al. 2011). If cattle are pregnant, then interferon tau released by the

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trophoblast prevents the development of the luteolytic mechanism by inhibiting expression of

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the oxytocin receptor gene in the endometrial epithelium, which prevents the release of

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luteolytic pulses of prostaglandin F2α (Dorniak, et al. 2013). Whereas, in the absence of an

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embryo, prostaglandin F2α induces luteolysis with the assistance of other inflammatory

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mediators such as TNFα (Skarzynski and Okuda 2010).

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Parturition and the postpartum period

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Progesterone maintains uterine quiescence throughout gestation but prior to parturition the

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influence of progesterone rapidly diminishes. In several species, the reduction in the influence

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of progesterone is associated with an increase in inflammatory mediators, such as IL-1β, IL-6,

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IL-8 and TNF-α, and an influx of leukocytes (Challis, et al. 2009). Similar mechanisms are

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likely important in cattle as there is increased expression of genes such as CXCL8, CXCL2

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and CXCL6 in placentomes of healthy cattle during parturition (Streyl, et al. 2012). Another

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feature of the peripartal bovine uterus is the accumulation of CD14+ monocytes and

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macrophages in the caruncular stroma and septa, although not in the foetal part of the

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placentomes (Miyoshi and Sawamukai 2004, Oliveira and Hansen 2009). Activated

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macrophages enhance the local inflammatory response by attracting neutrophils and

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monocytes, which clear apoptotic cells, and remodel the caruncle, aiding the successful

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release of the foetal membranes. Whilst the distribution and concentration of CD14+ cells is

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similar in healthy cows and cows that develop retained foetal membranes, the latter have

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lower concentrations of acid phosphatase, a lysosomal enzyme of macrophages, (Miyoshi, et

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al. 2002). The uterus must recover rapidly after parturition in preparation for establishment of

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the next pregnancy. Recovery involves tissue repair, regeneration of the epithelium lining the

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endometrium, and elimination of bacteria that always contaminate the uterus around the time

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of parturition.

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Microbial infections of the female reproductive tract

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Escherichia coli infect the endometrium within a week of parturition, followed in subsequent

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weeks by Trueperella pyogenes and a range of anaerobic bacteria that include Fusobacterium,

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Prevotella and Bacteriodes species (Bonnett, et al. 1991, Sheldon, et al. 2002, Sheldon, et al.

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2010). Although not the focus of the present manuscript, it is important to note that bovine

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herpesvirus 4 is also tropic for endometrial stromal cells, and causes postpartum uterine

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disease in some animals (Donofrio, et al. 2007). The pathogens most consistently associated

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with uterine disease have been established by traditional microbiological culture methods.

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However, using molecular techniques, many more non-culturable microbes are identified in

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the uterus of postpartum cattle, with or without clinical signs of uterine disease (Santos and

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Bicalho 2012). Indeed, molecular markers for some of these species of bacteria may lead to

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diagnostic tests for a healthy or diseased uterus. The presence of several acid-producing

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strains of bacteria in the uterine lumen is interesting because Lactobacilli predominate in the

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healthy human vaginal microbiome, where they help maintain a low pH to protect against

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pathogenic bacteria (Ravel, et al. 2011).

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Pathogens have evolved many virulence factors that may be important in the female

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reproductive tract, including toxins, proteases, neuramidases, secretion systems, fimbriae and

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iron acquisition systems. For example, endometrial pathogenic E. coli (EnPEC) adhere to and

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invade primary endometrial epithelial and stromal cells, cause inflammation, and recreate

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disease when infused into the uterus of mice (Sheldon, et al. 2010). In particular, LPS appears

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to be the major virulence factor of EnPEC, stimulating TLR4-dependent inflammatory

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responses by endometrial cells (Cronin, et al. 2012, Sheldon, et al. 2010). On the other hand,

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the presence of T. pyogenes is associated with pus in the uterus, the severity of clinical signs,

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and the extent of the subsequent subfertility (Bonnett, et al. 1991, Westermann, et al. 2010).

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The major virulence factor produced by all isolates of T. pyogenes is pyolysin, which is a

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heat-labile exotoxin that mulitmerises in cholesterol-rich domains of the plasma membrane of

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mammalian cells creating 30-50 nm diameter transmembrane pores to disrupt ion balances

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and cause cytolysis (Amos, et al. 2014, Jost and Billington 2005). Endometrial stromal cells

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are more sensitive to cytolysis by pyolysin than epithelial or immune cells, which explains

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how the bacteria act as pathogens, once the relatively resistant epithelium is denuded after

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parturition (Amos, et al. 2014). Interestingly, the higher cholesterol content of stromal cells,

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compared with epithelial cells, explains the sensitivity of stromal cells to pyolysin, and

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reducing cellular cholesterol using cycoldextrin protects stromal cells against pyolysin

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(Amos, et al. 2014).

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Animal models of uterine infection and disease

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The fundamental mechanisms of immunity and impact on the brain are most often studied by

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infusion of LPS into the uterus or into the peripheral circulation (Karsch, et al. 2002, Peter, et

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al. 1989). However, animal models of clinical uterine disease are also important because they

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are needed for development of new vaccines or treatments, as well as uncovering the

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mechanisms of immunity in the female reproductive tract. Animal models of uterine disease

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have been generated in dairy and beef cattle, sheep and mice (Amos, et al. 2014, Ayliffe and

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Noakes 1982, Karsch, et al. 2002, Sheldon, et al. 2010). Most of the animal models that

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replicate uterine disease rely on infusion of E. coli or T. pyogenes into the uterine lumen

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(Amos, et al. 2014, Ayliffe and Noakes 1982, Rowson, et al. 1953, Sheldon, et al. 2010). A

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key step in generating models of uterine disease in cattle is mechanical debridement of the

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epithelium lining the endometrium (Amos, et al. 2014, Ayliffe and Noakes 1982). Presumably

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this allows T. pyogenes and pyolysin to access the stromal compartment of the endometrium

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to establish infection and cause tissue damage. The duration and severity of clinical

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endometritis following infusion of bacteria can be increased by administering exogenous

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progesterone to the animal, or by infusion of bacteria during the luteal phase of the oestrous

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cycle (Lewis 2003, Rowson, et al. 1953). Similarly, early ovulation after parturition and

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establishment of a progesterone-dominated phase before pathogenic bacteria are eliminated

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predisposes to the development of pyometra (Olson, et al. 1984). Conversely, administering

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oestrogens or oestrus often precludes the establishment of uterine disease (Lewis 2003,

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Rowson, et al. 1953). However, the mechanisms linking the steroids to immunity are yet to

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be fully elucidated and are complex because there are interactions between the physiological

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effects of ovarian steroids and the inflammation caused by infusion of bacteria or LPS

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(Karsch, et al. 2002, Lewis 2003, Peter, et al. 1989).

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Endometrial responses to bacteria in the uterus

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Host responses to bacterial infection of the uterus are characterized by inflammation of the

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endometrium with infiltration by neutrophils and macrophages, and the accumulation of pus

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in the uterine lumen (Fig. 2). The diseases are defined as metritis, clinical endometritis, or

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subclinical endometritis depending on the signs and the time after parturition (Sheldon, et al.

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2006). Samples collected from the endometrium of diseased animals, compared with

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unaffected animals, have more abundant gene transcripts for: cytokines IL1A, IL1B, IL6 and

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TNF; chemokines CXCL8, CCL5 and CXCL5; calcium-binding proteins S100A8, S100A9 and

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S100A12; and receptors TLR4 and IL1R (Fischer, et al. 2010, Gabler, et al. 2009, Herath, et al.

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2009b, Wathes, et al. 2009). Furthermore, there are temporal changes in the expression of

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transcripts for IL1B, IL6, CXCL8, TNF, CXCL5, HP and PTGS2 during the postpartum

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period, with peak expression on day 17 after parturition (Gabler, et al. 2010). These temporal

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changes in gene expression reflect the histological evidence of inflammation during the first 3

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weeks post partum, which resolves by 40 to 60 days after parturition (Bonnett, et al. 1991,

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Chapwanya, et al. 2012). As well as increased gene expression, postpartum cows, particularly

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if the endometrium is infected, have increased protein abundance of S100A8 and S100A9 in

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the

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immunohistochemistry (Swangchan-Uthai, et al. 2013). The findings at the transcription level

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are also supported by analysis of the proteome profile of cows with endometritis, which have

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more S100A9 and cathelicidin antimicrobial protein in the endometrium, compared with

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healthy cows (Ledgard, et al. 2013). Taken together, these endometrial inflammatory

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responses are likely part of the physiological mechanisms of tissue homeostasis (Medzhitov

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2008). However, stressors such as limitations on nutrient supply around the time of parturition

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may lead to excessive inflammatory responses and increased risk of disease (Swangchan-

336

Uthai, et al. 2013, Wathes, et al. 2009).

epithelium

and stroma,

as

well as

in

immune

cells,

as

determined

by

337 338

The tissue responses following postpartum bacterial infection point to an important role for

339

innate immunity. Endometrial biopsies collected from healthy and diseased postpartum dairy

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cows express mRNA for all ten TLRs (Herath, et al. 2009b). In addition, ex vivo organ

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cultures of bovine endometrium accumulate IL-1β, IL-6, IL-8, and prostaglandin E2 when

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challenged with E. coli or T. pyogenes (Amos, et al. 2014, Borges, et al. 2012). Furthermore,

343

pure populations of endometrial epithelial and stromal cells express most TLRs, and their co-

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receptors, such as MD2 and CD14 (Davies, et al. 2008, Herath, et al. 2006). Addition of LPS

345

to epithelial or stromal cells in vitro stimulates phosphorylation of MAPK and the nuclear

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translocation of NFκB, secretion of prostaglandin F2α, prostaglandin E2, IL-6 and IL-8, and

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increased expression of antimicrobial peptides MUC1, LAP and TAP (Cronin, et al. 2012,

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Davies, et al. 2008, Herath, et al. 2006). Intriguingly, the release of IL-8 from polarised

349

epithelial cells is vectorial and directed apically if the challenge is in the uterine lumen

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(MacKintosh, et al. 2013). Similar cytokine and chemokine responses are also evident for

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cells treated with bacterial lipopeptides, with formal proof for the importance of innate

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immunity established using siRNA targeting TLR1, TLR2, TLR4 and TLR6 to suppress

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inflammation (Cronin, et al. 2012, Turner, et al. 2014). There are also increased peripheral

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blood concentrations of acute phase proteins such as serum amyloid A and haptoglobin,

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which are produced by hepatocytes in response to cytokines such as IL-6 from the infected

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uterus (Sheldon, et al. 2001). Taken together these data support the concept that the epithelial

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and stromal cells in the endometrium have roles in the initial sensing and response to

358

pathogens (Fig. 2). A remaining question is the role of DAMPs in endometritis because

359

uterine infections are often associated with tissue damage including dystocia, retained foetal

360

membranes, and a large male calf.

361 362

Ovarian follicle responses to bacteria in the uterus

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Follicle and oocyte development is a highly coordinated series of events starting with

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primordial to primary and secondary follicle transition, controlled by autocrine and paracrine

365

signals including PTEN, c-kit and FOXO3 (Castrillon, et al. 2003, Parrott and Skinner 1999,

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Reddy, et al. 2008). This is followed by gonadotrophin dependent follicle growth, before

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formation of the follicle antrum and then selection of the dominant follicle. Whilst links

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between inflammation in the endometrium and infertility are intuitive, it was less obvious

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whether uterine infections might affect ovarian function. However, in dairy cattle, uterine

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disease is associated with LPS in follicular fluid, slower growth of dominant follicles, lower

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peripheral plasma concentrations of oestradiol, and increased risks of anoestrus or cystic

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ovarian disease (Herath, et al. 2007, Opsomer, et al. 2000, Sheldon, et al. 2002). The

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mechanisms underlying these observations are now emerging.

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During early bovine ovarian follicle development, LPS increases ex vivo primordial to

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primary follicle activation, which is associated with loss of primordial follicle PTEN and

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cytoplasmic translocation of FOXO3 (Bromfield and Sheldon 2013). The implication of this

378

work is that multiple bacterial infections could deplete the ovarian follicle reserve; although,

379

LPS does not affect secondary follicle growth and function (Bromfield and Sheldon 2013).

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Antral follicles present a special situation because whilst the stroma of the ovary contains a

381

wide range of immune cells including macrophages that might protect pre-antral follicles,

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healthy antral follicles are devoid of such cells within the basement membrane (Bromfield

383

and Sheldon 2011). Intriguingly, mural granulosa cells lining emerged and dominant antral

384

follicles express TLRs and secrete IL-1β, IL-6 and IL-8 when treated with ligands that bind

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TLR2 or TLR4 (Bromfield and Sheldon 2011, Price, et al. 2013, Price and Sheldon 2013).

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Treatment with PAMPs also reduces the expression of CYP19A1 mRNA and aromatase

387

protein, as well as the secretion of oestradiol from granulosa cells (Herath, et al. 2007, Price,

388

et al. 2013). Granulosa cell responses are rapid, with increased phosphorylation of p38 and

389

ERK1/2, and expression of IL6, IL1B, IL10, TNF, CXCL8 and CCL5 mRNA (Bromfield and

390

Sheldon 2011, Price, et al. 2013, Price and Sheldon 2013). Furthermore, the innate immune

391

response is attenuated using siRNA targeting TLR2 or TLR4, or by treating granulosa cells

392

with inhibitors targeting MAPK or NFκB.

393 394

As physiological events within the ovary use several inflammatory mediators, events

395

associated with infection of the female genital tract may disrupt the biology of reproduction.

396

For example, expansion of the cumulus-oocyte complex is a key event during ovulation but,

397

even in the absence of gonadotropin, high concentrations of LPS cause inappropriate

398

expansion, which would compromise fertility (Bromfield and Sheldon 2011). Even the oocyte

399

is at risk from pathogens, and BVD and IBR are amongst viruses that have been isolated from

15

Page 16 of 37

400

oocytes denuded of granulosa cells. Furthermore, in vitro maturation of oocytes in the

401

presence of LPS, reduces meiotic competence from 85% to 64%, and this failure cannot be

402

rescued by the administration of exogenous gonadotrophins (Bromfield and Sheldon 2013).

403

So, alterations in the follicular environment due to pathogen exposure results in diminished

404

oocyte quality, further affecting fertility.

405 406

Host responses in the brain associated with uterine disease

407

In postpartum cows, LPS in the uterus suppresses the luteinising hormone (LH) surge and

408

prevents ovulation (Peter, et al. 1989). Also, during the follicular phase LPS decreases LH

409

pulse frequency, decreases oestradiol and interrupts the LH surge and ovulation in cattle

410

(Lavon, et al. 2008, Suzuki, et al. 2001). Similar observations have been made in sheep,

411

which is the species most often used to study the effect of LPS in the ruminant brain

412

(reviewed by Karsch et al 2002). Neuroendocrine activity at the hypothalamic level is

413

inhibited by LPS suppressing both the frequency and amplitude of GnRH/LH pulses.

414

However, there are also effects within the pituitary gland as LPS lowers LH responses to

415

exogenous GnRH (Karsch, et al. 2002). Indeed, treatment of ewes with LPS suppresses the

416

expression of genes encoding GnRH receptor (GnRHR) and LHβ (Herman, et al. 2013b).

417

However, the peripheral plasma concentrations of follicle stimulating hormone are unaffected

418

by uterine disease in cattle (Sheldon, et al. 2002).

419 420

The mechanisms linking LPS administration to changes in the hypothalamus and pituitary are

421

elusive. One possibility is that increased circulating cortisol, associated with the stress of LPS

422

treatment, modulates brain function. However, whilst exogenous cortisol suppresses pulsatile

423

LH secretion in ewes, LPS still inhibits GnRH and LH pulsatility when endogenous cortisol

424

secretion is inhibited (Karsch, et al. 2002). Opioids are implicated in LPS suppression of LH

16

Page 17 of 37

425

because an opioid receptor antagonist increases LH responses to LPS in heifers (Kujjo, et al.

426

1995). However, the same opioid receptor antagonist does not alter the reduction in LH in

427

response to acute bacteraemia (Leshin and Malven 1984). A similar conundrum is evident

428

using inhibitors of prostaglandin synthesis, which counter the LPS-induced suppression of LH

429

pulses but not the LPS-induced blockage of the LH surge (Breen, et al. 2004, Karsch, et al.

430

2002). Another approach to understanding how LPS affects hypothalamic activity is to

431

consider innate immunity. Preliminary results show that LPS increases the expression of

432

TLR4 genes in the medial preoptic area (mPOA), anterior hypothalamus, medial basal

433

hypothalamus (comprising the arcuate nucleus, ARC, and ventromedial nucleus, VMN) and

434

median eminence (Fig 3) (Herman, et al. 2013a). Peripheral LPS also increases FOS and IL1B

435

mRNA in the ventricular system, which is the choroid plexus in the brain endothelium

436

(Vellucci, et al. 1995); and administration of IL-1β into the ventricular system suppresses

437

GNRHR and LHB gene expression in the sheep pituitary (Herman, et al. 2012). Therefore,

438

LPS and inflammatory mediators may act centrally to inhibit neuroendocrine events;

439

although, there needs to be some caution because IL-1β and TNFα also act as normal

440

neuromodulators within the hypothalamus.

441 442

Administration of LPS during the follicular phase causes a delay in LH surge onset and this is

443

positively correlated with the duration of oestradiol signal disruption (Fergani, et al. 2012).

444

Also, the expression of GNRH and GNRHR mRNA in the mPOA and median eminence is

445

lower, but unexpectedly not in the medial basal hypothalamus (Herman, et al. 2012). These

446

neuroendocrine disruptions are associated with an increase in c-Fos within the VMN, ARC,

447

mPOA and diagonal band of Broca but there is no anticipated activation of oestradiol receptor

448

(ERα) containing cells in the ARC and mPOA (Fig 3) (Fergani, et al. 2012). LPS also inhibits

449

kisspeptin and dynorphin cell activation in the ARC and kisspeptin in the mPOA, and

17

Page 18 of 37

450

increases somatostatin activation in the VMN (Fergani, et al. 2013); and unpublished data).

451

Somatostatin is one of the most potent inhibitors of GnRH neurone excitability so far

452

identified (Bhattarai, et al. 2010).

453 454

Not surprisingly, LPS treatment increases FOS and corticotrophin releasing factor (CRF)

455

mRNA within the paraventricular nucleus, the stress control centre of the hypothalamus

456

(Vellucci, et al. 1995) as well as increasing CRF receptors (CRFR) in the ARC and median

457

eminence (Fig. 3) (Fergani, et al. 2013). Therefore, there are two possible pathways for CRF

458

suppression of GnRH, one being the direct association of CRF and GnRH cell terminals in the

459

ME (Ghuman, et al. 2010), and the other being the regulation of kisspeptin-neurokinin B-

460

dynorphin cells and others in the ARC and median eminence (Fergani, et al. 2013).

461 462

A cholinergic pathway is also involved in restraining the central response to LPS. Peripheral

463

LPS increases IL1B and IL1R1 gene expression as well as IL-1β concentration in the mPOA

464

and the hypothalamus, but the effect is reversed by concomitant rivastigmine, an

465

acetylcholinesterase inhibitor (Herman, et al. 2012). Indeed, acetylcholine itself reduces LPS-

466

induced release of IL-1β and TNFα. Furthermore, rivastigmine reverses LPS-induced

467

suppression GnRH and GnRH-R mRNA in the ME as well as LHB and GNRHR mRNA in the

468

pituitary (Herman, et al. 2013b).

469 470

A final caveat is that many of the GnRH/LH responses are not specific to LPS. Similar

471

changes occur following other stressors, for example, shearing, transport, isolation (Dobson,

472

et al. 2012). Thus, future hypotheses must take into account similarities and differences in

473

responses, as well as issues relating to stressors of differing intensity.

474

18

Page 19 of 37

475

Conclusions

476

The integration of innate immunity and inflammation in the female reproductive system has

477

likely evolved over millennia. There appears to be adoption of inflammatory mediators in the

478

physiology of reproduction in cattle, whilst innate immunity remains important for

479

maintenance of tissue homoeostasis and defence against pathogens. In dairy cattle, the need to

480

control postpartum infections of the genital tract highlights the importance of understanding

481

the mechanisms underlying the integration of innate immunity, inflammation and

482

reproductive physiology. In addition, these interactions are modulated by stress, the

483

environment, and metabolism, which are likely to become more important with the need to

484

feed the expanding human population and to counter climate change. An integrated approach

485

to understanding the interactions between innate immunity, inflammation, and reproduction

486

will be important for developing strategies to maintain animal health and welfare

487 488 489 490 491 492 493 494 495 496 497 498 499

19

Page 20 of 37

500

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of foetal membranes. Reproduction 143 85-105.

755

Suzuki, C, K Yoshioka, S Iwamura, and H Hirose 2001 Endotoxin induces delayed

756

ovulation following endocrine aberration during the proestrous phase in Holstein

757

heifers. Domestic Animal Endocrinology 20 267-278.

758

Swangchan-Uthai, T, Q Chen, SE Kirton, MA Fenwick, Z Cheng, J Patton, AA Fouladi-

759

Nashta, and DC Wathes 2013 Influence of energy balance on the antimicrobial

760

peptides S100A8 and S100A9 in the endometrium of the post-partum dairy cow.

761

Reproduction 145 527-539.

762 763

Takeuchi, O, and S Akira 2010 Pattern recognition receptors and inflammation. Cell 140 805-820.

764

Turner, ML, JC Cronin, GD Healey, and IM Sheldon 2014 Epithelial and stromal cells

765

of bovine endometrium have roles in innate immunity and initiate inflammatory

766

responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2, TLR1

767

and TLR6. Endocrinology 155 1453-1465

768 769

Turner, ML, GD Healey, and IM Sheldon 2012 Immunity and inflammation in the uterus. Reproduction in Domestic Animals 47 Suppl 4 402-409.

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770

Vellucci, SV, RF Parrott, AC da Costa, S Ohkura, and KM Kendrick 1995 Increased

771

body temperature, cortisol secretion, and hypothalamic expression of c-fos,

772

corticotrophin releasing hormone and interleukin-1 beta mRNAs, following

773

central administration of interleukin-1 beta in the sheep. Brain Research 29 64-

774

70.

775

Wathes, DC, Z Cheng, W Chowdhury, MA Fenwick, R Fitzpatrick, DG Morris, J

776

Patton, and JJ Murphy 2009 Negative energy balance alters global gene

777

expression and immune responses in the uterus of postpartum dairy cows.

778

Physiolical Genomics 39 1-13.

779

Westermann, S, M Drillich, TB Kaufmann, LV Madoz, and W Heuwieser 2010 A

780

clinical approach to determine false positive findings of clinical endometritis by

781

vaginoscopy by the use of uterine bacteriology and cytology in dairy cows.

782

Theriogenology 74 1248-1255.

783 784 785 786 787 788 789 790 791 792 793 794

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Page 32 of 37

795

Figure Legends

796 797

Figure 1 Signalling pathways of the Toll-like Receptors

798

During infection PAMPs initiate signalling pathways by interacting with TLRs, resulting in

799

formation of homo- or heterodimers and activation of cytosolic Toll/IL-1 receptor (TIR)

800

domains. Downstream signalling pathways are initiated through four adaptor proteins:

801

MyD88; Mal, TRIF, or TRAM. All TLRs, with the exception of TLR3, use the MyD88-

802

dependent pathway. Mal acts as a bridge to recruit MyD88 to TLR2 and TLR4 signalling. The

803

adaptor TRIF is used in TLR3 signalling and, in association with TRAM, in endosomal TLR4

804

signalling. In the MyD88-dependent pathway, MyD88 associates with IRAK4, IRAK1

805

(and/or IRAK2; not illustrated). IRAK4 in turn phosphorylates IRAK1 and promotes

806

association with TRAF6, which serves as a platform to recruit and activate the kinase TAK1.

807

Activated TAK1 leads to the activation of the MAPK cascade. The MAPKs ERK, JNK and

808

p38 enhance the expression of pro-inflammatory cytokines via activation of AP-1 (for

809

simplicity, only activation of AP-1 is illustrated in the diagram). TAK1 also leads to

810

activation of the IKK complex, composed of IKKα, IKKβ, and IKKγ, which results in the

811

phosphorylation and ubiquitination of IκB. Ubiquitination results in degradation of IκB,

812

freeing NFκB to translocate from the cytoplasm to the nucleus, where it activates gene

813

expression in concert with AP-1. Endosomal TLR3 and TLR4 signalling, through the adaptor

814

protein TRIF, leads to activation of TBK1 or IKKε, inducing IRF3 dimerization and

815

translocation into the nucleus where it induces transcription of type I IFN and IFN-inducible

816

genes, including IFNβ. Downstream of TLR7, TLR8, and TLR9, IRF7 is directly

817

phosphorylated by IRAK1 and translocates into the nucleus to induce the expression of type I

818

IFN and IFN-inducible genes, including IFNα.

819

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Page 33 of 37

820

Figure 2 Pathogenic bacteria cause inflammation in the endometrium

821

After parturition, Escherichia coli, Trueperella pyogenes and other bacteria invade the uterus,

822

and the pathogenic bacteria adhere to and invade the endometrium, particularly where there is

823

tissue damage. Truperella pyogenes also produces pyolysin, which creates transmembrane

824

pores in endometrial cells, causing cell death and further tissue damage. Endometrial

825

epithelial and stromal cells sense pathogen-associated and damage-associated molecular

826

patterns via innate immune receptors, such as TLRs, which leads to the release of cytokines

827

and chemokines, such as IL-1B, IL-6 and IL-8 (black arrows). These inflammatory mediators

828

attract and activate hematopoietic cells, particularly neutrophils and macrophages, to the site

829

of infection (red arrow), in order to clear the invading microbes and resolve the tissue

830

damage.

831 832

Figure 3 Interaction between neurones involved in reproduction, behaviour and stress

833

Noradrenergic cells in the ovine brain stem project to both the preoptic area (reproduction)

834

and the paraventricular nucleus (stress centre); LPS activates neurones in the latter. Changes

835

in activity of β-endorphin and dynorphin neurones of the arcuate nucleus influence the

836

paraventricular nucleus and medial preoptic area output. In the arcuate nucleus, the activities

837

of oestradiol receptor (ER)α-positive neurones (kisspeptin/dynorphin/neurokinin B cells) are

838

modulated by LPS, as are the somatostatin cells in the ventromedial nucleus. In the median

839

eminence, corticotrophin releasing hormone (CRH), but not arginine vasopressin (AVP)

840

terminals, and kisspeptin/dynorphin/neurokinin B terminals are in close contact with GnRH

841

terminals, possibly providing another site for the disruption of GnRH release; the normal

842

profile of GnRH release (dotted line) is suppressed during stress (grey infill). Neural

843

activation and suppressive influences are indicated by + and - symbols, respectively. Adapted

844

from original drawing by SPS Ghuman and reproduced with permission.

33

Page 34 of 37

Table 1 Pattern recognition receptors, their localisation, and the pathogen-associated molecular patterns they bind Receptor

Localization

Pathogen-associated molecular patterns

TLR1

Plasma Membrane

Triacylated bacterial lipopeptides

TLR2

Plasma Membrane

Bacterial lipopeptides, glycolipids, peptidoglycan

TLR3

Viral double-stranded RNA

TLR5

Endosome Plasma Membrane/Endosome Plasma Membrane

TLR6

Plasma Membrane

Diacylated bacterial lipopeptides

TLR7

Endosome

Viral and bacterial single-stranded RNA

TLR8

Endosome

Viral and bacterial single-stranded RNA

TLR9

Endosome

CpG-rich bacterial and viral DNA

TLR10

Plasma Membrane

Unknown

RIG-I

Cytoplasm

Short double stranded RNA

NOD1

Cytoplasm

D-glutamyl-meso-diaminopimelic acid

NOD2

Cytoplasm

Muramyl dipeptide

NLRP3

Cytoplasm

Uric acid crystals, cholesterol-dependent cytolysins

NLRC4

Cytoplasm

Flagellin, type III secretion system proteins

TLR4

Lipopolysaccharide Flagellin

Page 35 of 37

Page 36 of 37

IL-6

IL-1b

IL-1b IL-1b

IL-8

IL-6

IL-1b

IL-6 IL-8

Epithelium

IL-8

Stroma

Capillary

E. coli IL-1b

IL-1b

T. pyogenes IL-6

IL-6

IL-8

Pyolysin pore

Neutrophil

IL-8

Macrophage

Noradrenergic

Page 37 of 37

ER ER

Reproduction _ Medial preoptic area

_ GnRH _ _ _

_

Paraventricular nucleus

ER

GABA

+ AVP

+ Dopamine

+

Ventromedial nucleus

ER ER

_+

+

Caudal brainstem

ER ER

_

CRH

_

Stress LPS

_ _

GABA

GABA

+

ER

ER

Somatostatin

Arcuate? nucleus

Arcuate nucleus ER

Dopamine

_

ER

Kisspeptin Dynorphin Neurokinin B

AVP

_ ER

Kisspeptin Dynorphin Neurokinin B

Behaviour + _ GnRH

GABA

Median eminence Portal blood

Innate immunity and inflammation of the bovine female reproductive tract in health and disease.

Mammalian reproductive physiology and the development of viviparity co-evolved with inflammation and immunity over millennia. Many inflammatory mediat...
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