David J. Sharkey

Transforming growth factor β—a mediator of immune deviation in seminal plasma

TGFβ is a potent immune deviating agent, driving active forms of immune tolerance in peripheral tissues through effects on the induction and resolution of inflammatory responses and phenotype skewing in antigen-presenting cells and lymphocytes. The TGFβ content of seminal plasma from human, rodent and livestock species is amongst the highest measured in biological fluids. The seminal vesicle gland is the principal source of TGFβ in the semen of mice, where its synthesis is regulated by testosterone. At insemination, seminal TGFβ is deposited in the female tract and is activated by acidic vaginal pH, enzymes of male or female tract origin, or through cleavage-independent processes involving conformational change after interaction with epithelial cell docking proteins. Seminal TGFβ has been shown to be a principal stimulating agent in the post-coital inflammatory response, and is likely to be essential for induction of immune tolerance to seminal antigens. As well as preventing aberrant immunity to spermatozoa, these events are implicated in priming an appropriate female immune response to embryo implantation, since many seminal antigens are shared by the conceptus. The cascade of immunological events elicited by seminal TGFβ may therefore explain epidemiological observations linking acute and cumulative exposure to semen with successful placental development and pregnancy outcome. Depending on whether the female tract has evolved mechanisms to discriminate seminal antigens from opportunistic pathogens, there may be a detrimental cost of seminal TGFβ in inhibiting protective immunity to agents of sexually transmitted disease including HIV. A better understanding of the significance and role of TGFβ in semen will facilitate development of novel therapies for immune-based infertility disorders.

Seminal Fluid Induces Leukocyte Recruitment and Cytokine and Chemokine mRNA Expression in the Human Cervix after Coitus

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner’s seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45+ cells mainly comprising CD14+ macrophages and CD1a+ dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3+CD8+CD45RO+ T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.

TGF-β Mediates Proinflammatory Seminal Fluid Signaling in Human Cervical Epithelial Cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner’s seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Seminal Fluid and the Generation of Regulatory T Cells for Embryo Implantation

T regulatory (Treg) cells are essential mediators of the maternal immune adaptation necessary for embryo implantation. In mice, insufficient Treg cell activity results in implantation failure, or constrains placental function and fetal growth. In women, Treg cell deficiency is linked with unexplained infertility, miscarriage, and pre‐eclampsia. To devise strategies to improve Treg cell function, it is essential to define the origin of the Treg cells in gestational tissues, and the regulators that control their functional competence and recruitment. Male seminal fluid is a potent source of the Treg cell‐inducing agents TGFβ and prostaglandin E, and coitus is one key factor involved in expanding the pool of inducible Treg cells that react with paternal alloantigens shared by conceptus tissues. In mice, coitus initiates a sequence of events whereby female dendritic cells cross‐present seminal fluid antigens and activate T cells, which in turn circulate via the blood to be sequestered into the endometrium. Similar events may occur in the human genital tract, where seminal fluid induces immune cell changes that appear competent to prime Treg cells. Improved understanding of how seminal fluid influences Treg cells in women should ultimately assist in the development of new therapies for immune‐mediated pathologies of pregnancy.

TLR4 Signaling Is a Major Mediator of the Female Tract Response to Seminal Fluid in Mice

ABSTRACT Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

Hormonal regulation of the immune microenvironment in the mammary gland.

The mammary gland is a unique organ that undergoes hormone-driven developmental changes over the course of the ovarian cycle during adult life. Macrophages play a role in regulating cellular turnover in the mammary gland and may affect cancer susceptibility. However, the immune microenvironment that regulates macrophage function has not been described. Hormonal regulation of the cytokine microenvironment across the ovarian cycle was explored using microbead multiplex assay for 15 cytokines in mammary glands from C57Bl/6 mice at different stages of the oestrous cycle, and in ovariectomised mice administered oestradiol and progesterone. The cytokines that were found to fluctuate over the course of the oestrous cycle were colony-stimulating factor (CSF)1, CSF2, interferon gamma (IFNG) and tumour necrosis factor alpha (TNFA), all of which were significantly elevated at oestrus compared with other phases. The concentration of serum progesterone during the oestrus phase negatively correlated with the abundance of cytokines CSF3, IL12p40, IFNG and leukaemia inhibitory factor (LIF). In ovariectomised mice, exogenous oestradiol administration increased mammary gland CSF1, CSF2, IFNG and LIF, compared with ovariectomised control mice. Progesterone administration together with oestradiol resulted in reduced CSF1, CSF3 and IFNG compared with oestradiol administration alone. This study suggests that the cytokine microenvironment in the mammary gland at the oestrus phase of the ovarian cycle is relatively pro-inflammatory compared with other stages of the cycle, and that the oestradiol-induced cytokine microenvironment is significantly attenuated by progesterone. A continuously fluctuating cytokine microenvironment in the mammary gland presumably regulates the phenotypes of resident leukocytes and may affect mammary gland cancer susceptibility.

Molecular Filtration Properties of the Mouse Expanded Cumulus Matrix: Controlled Supply of Metabolites and Extracellular Signals to Cumulus Cells and the Oocyte

ABSTRACT While formation of the expanded cumulus matrix and its importance for oocyte maturation and ovulation are well described, its function in these processes remains unknown. The degree of expansion and expression of cumulus matrix genes are positively correlated with oocyte quality, suggesting that this matrix plays a key role in oocyte maturation. Based on recognized filtration properties of analogous matrices, we investigated whether the cumulus matrix acts as a molecular filter by assessing diffusion of fluorescently labeled dextrans (neutral and negatively charged) and hydrophilic (glucose) and hydrophobic (cholesterol) metabolites in cumulus oocyte complexes (COCs). Expanded in vivo-matured COCs resisted absorption of glucose and cholesterol compared to unexpanded COCs. In vitro-matured (IVM) COCs have a pronounced deficiency in cumulus matrix proteins and have poor oocyte quality. Here we demonstrate that IVM cumulus matrix has deficient filtration properties, with dextran and glucose and cholesterol molecules diffusing more readily into IVM than in vivo-matured COCs. Taking the inverse approach, we found that prostaglandin E2 (PGE2), synthesized by cumulus cells, is retained within the matrix of in vivo-matured COCs but IVM COCs have reduced capacity to retain PGE2, secreting significantly more into the medium. This is the first demonstration of a biophysical property of the cumulus matrix. The ability to regulate metabolite supply from the surrounding environment while sequestering vital signaling factors, such as PGE2, is likely to impact oocyte maturation. Thus, IVM may reduce oocyte quality due to dysregulated control of metabolites and signaling molecules.

Antenatal Suppression of IL-1 Protects against Inflammation-Induced Fetal Injury and Improves Neonatal and Developmental Outcomes in Mice

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1β induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1β, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1β, IL-6, and IL-8, increased IL-1β, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.

miRNA Regulation of Immune Tolerance in Early Pregnancy

To support embryo implantation, the female reproductive tract must provide a tolerogenic immune environment. Seminal fluid contact at conception contributes to activating the endometrial gene expression and immune cell changes required for robust implantation, influencing not only the quality of the ensuing pregnancy but also the health of offspring. miRNAs are small non‐coding RNAs that play important regulatory roles in biological processes, including regulation of the immune environment. miRNAs are known to contribute to gene regulation in pregnancy and are altered in pregnancy pathologies. Recent studies indicate that miRNAs participate in establishing immune tolerance at conception, and may contribute to the regulatory effects of seminal fluid in generating tolerogenic dendritic cells and T regulatory cells. This review highlights those miRNAs implicated in programming immune cells that are critical during the peri‐conception period and explores how seminal fluid may regulate female tract miRNA expression following coitus.

Seminal plasma transforming growth factor-β, activin A and follistatin fluctuate within men over time.

STUDY QUESTION Do seminal plasma transforming growth factor-β (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? SUMMARY ANSWER Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. WHAT IS ALREADY KNOWN Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. STUDY DESIGN, SIZE, DURATION Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. PARTICIPANTS/MATERIALS, SETTING AND METHOD The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. LIMITATIONS, REASONS FOR CAUTION A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individuals status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.