Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFß, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.

The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization.

LABELED PROBLEM: Embryo implantation remains the main limiting factor in assisted reproductive medicine (20% success rate). METHODS OF STUDY: An endometrial immune profiling was performed among 394 women with the previous history of repeated embryo implantation failures (RIF). The endometrial immune profile documented the ratio of IL-15/Fn-14 mRNA as a biomarker of uNK cell activation/maturation (together with the uNK cell count) and the IL-18/TWEAK mRNA ratio as a biomarker of both angiogenesis and the Th1/Th2 balance. According to their profile, we recommended personalized care to counteract the documented dysregulation and assessed its effects by the live birth rate (LBR) for the next embryo transfer. RESULTS: Endometrial immune profiles appeared to be dysregulated in 81.7% of the RIF patients compared to control. Overactivation was diagnosed in 56.6% and low activation in 25%. The LBR among these dysregulated/treated patients at the first subsequent embryo transfer was 39.8%. CONCLUSION: Endometrial immune profiling may improve our understanding of RIF and subsequent LBR if treated.

Colony Stimulating Factors 1, 2, 3 and early pregnancy steps: from bench to bedside.

Reproductive immunology applies general immunology principles to specialised targets, reproduction and development. The involvement of colony-stimulating factors (CSFs) in reproduction illustrates this. The CSF family includes CSF-1 or macrophage CSF (M-CSF), CSF-2 or granulocyte macrophage CSF (GM-CSF), and CSF-3 or granulocyte CSF (G-CSF). Each member has a specific localisation and timed expression in the reproductive tract with specific functions involving them in ovulation, embryo implantation, placentation and further embryonic development. They are used in reproductive medicine, either as biomarkers of oocyte quality and competence (follicular G-CSF), or to supplement embryo culture media with human recombinant GM-CSF, or they are used as an innovative therapy by using human recombinant G-CSF for infertile patients. Given fundamental considerations on CSFs and their strong implication in reproduction, this review aimed to detail the current knowledge for each member of the family to improve our understanding of their implication in the maternal-foetal cytokinic dialogue and in possibly preventing reproductive disorders.

Granulocyte-Colony Stimulating Factor related pathways tested on an endometrial ex-vivo model.

INTRODUCTION: Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. MATERIALS AND METHODS: Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. RESULTS: At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. CONCLUSION: RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early pregnancy on early embryogenesis still needs to be demonstrated. Nevertheless, rhG-CSF appears as a promising therapy in some difficult and unsolved cases of reproductive failure. Indications of pre-conceptual rhG-CSF supplementation may derive from a diagnosed lack of endometrial expression of some target genes.

An insight into normal and pathological pregnancies using large-scale microarrays: lessons from microarrays.

In the introduction, we briefly recall old but classic evidence that there is no tolerance to paternal alloantigens in a first pregnancy. Therefore, we performed small- and large-scale microarrays in CBA × DBA/2 and CBA × BALB/c combinations, recently described as a murine model for preeclampsia. Our results are in line with other data suggesting a very early deregulation of local immune vascular events rather than a break of immune tolerance. Other data presented at the Tioman 2010 Preeclampsia Workshop supporting this hypothesis are briefly summarised, as well as indications and caveats from a recent human microarray on implantation failure and recurrent pregnancy loss.

TWEAK appears as a modulator of endometrial IL-18 related cytotoxic activity of uterine natural killers.

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human.

Tolerance to the foetal allograft?

In this review, we will detail the concept of tolerance and its history in reproductive immunology. We will then consider whether it applies to the foetal-maternal relationship and discuss the mechanisms involved in non-rejection of the foeto-placental unit.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor inducible-14 might regulate the effects of interleukin 18 and 15 in the human endometrium.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) could play a role in the regulation of interleukin (IL)-15 and IL-18, cytokines crucial for angiogenesis and uterine natural killer (uNK) cell recruitment during embryo implantation. We therefore confirmed the endometrial presence of TWEAK/fibroblast growth factor inducible-14 (Fn-14) and documented simultaneously the cytotoxic KIR receptor (NKp46) of uNK cells in the human endometrium while TWEAK, Fn-14, IL-15, and IL-18 mRNA were quantified by real-time polymerase chain reaction in relation to the recruitment of CD56+ cells among fertile control women and patients who had failed to implant after assisted reproduction treatment.

Early regulators in abortion and implications for a preeclampsia model.

This paper reports a summary of our comparative analysis of the uterine expression of interleukin-23 (IL-23), IL-27 and TWEAK in the CBA/J femalexDBA/2 male mouse mating combination, a model of immune-mediated early pregnancy loss. Compared with the MHC-identical CBA/JxBALB/c mating combination, which yields successful pregnancies, immunohistochemistry and qPCR in uterine tissue showed an immediate post-mating IL-27 hyper-expression after mating with DBA/2 males. Intra-uterine TWEAK expression was present in females mated with DBA/2 or Balb/c males from days 0.5 to 4.5 post-coitum (pc), peaking on day 0.5 pc together with uterine TNFalpha. In uteri of DBA/2 mated mice, TWEAK declined to almost undetectable levels on days 6.5-9.5 pc, a steeper drop than in BALB/c mated mice where TWEAK remained detectable. In both mating combinations, neutralisation of TWEAK by antibodies increased resorption rates, but surprisingly, so did IL-27 neutralisation. The complement regulator mannan binding lectin-A (MBL-A), but not MBL-C, was present on day 4.5 pc especially after mating with DBA/2 males. High levels of MBL are present in the uterine luminal fluid of sterile women, and possible functions for TWEAK and MBL in human implantation are indicated by their protein and mRNA expression in uterine biopsies from infertile and fertile individuals. Consistent with the results in mice, increased MBL expression is linked with pregnancy failure. Serum and uterine VEGF and VEGF receptor levels are also different between fertile and sterile patients. The implications of these findings for utilising the CBA/JxDBA/2 mating combination as an early onset model of preeclampsia are discussed.

Immune regulation at the interface during early steps of murine implantation: involvement of two new cytokines of the IL-12 family (IL-23 and IL-27) and of TWEAK.

PROBLEM: An important subset of implantation defects/early abortion seems to be linked with a deregulation of the interleukin (IL)-12/IL-15/IL-18 system as well as tumor necrosis factor (TNF)- and natural killer (NK)-controlled/mediated networks at the decidual placental interface, both in case of deficient or excess expression. The presence of TNF in high amounts in the pre/peri-implantation uterus and its pivotal role during pregnancy are difficult to reconcile with its abortive effects in ongoing pregnancy. We therefore searched for regulators of the IL-12/IL-18 family of cytokines as well as for antagonist(s) of TNF with potentially selective effects on implantation. METHOD OF STUDY: We first used Swiss mice to verify the presence in the murine reproductive tract of 'new members' of the IL-12 family of cytokines, IL-23 and IL-27, as well as of tumor necrosis factor-like WEAK inducer of apoptosis (TWEAK), described as acting with TNF as Yin and Yang of innate immunity in murine placenta/ decidua at days 0-12.5. We then compared expression by RT-PCR in the CBA x DBA/2, and CBA x BALB/c murine mating combinations. Finally, we performed in vivo neutralization experiments of TWEAK and IL-27. RESULTS: Immunohistochemistry (IHC) studies showed that IL-23, IL-27, and TWEAK were expressed at the interface. For RT-PCR, IL-23 expression peaked at day 9.5 in the non-aborting mating combination, a peak absent in the aborting one, and thus difficult to explain except by invoking a feed back on EB13 (Epstein-Barr virus-induced gene 3 cytokine). Most important, an immediate post-mating IL-27 hyper expression was seen in the CBA x DBA/2 mating compared to CBA x BALB/c one. The difference in expression resurged and was statistically very significant by days 6.5-9.5, compatible with an early activation of inflammation on day 0.5 which would then peak again in the 'resorption window' where takes place the early NK/mph activation described by Baines et al. A significant TWEAK expression was present in both strains from days 0.5 to 4.5 peaking in both cases in the first days when it is known that intra uterine TNF also reaches high levels as a component of post-mating inflammation. However, it was lower from day 1.5 in the abortion-prone CBA x DBA/2 mating combination, and almost absent by days 6.5-9.5 when compared to the non-aborting CBA x BALB/c mating combination. In both mating combinations, neutralization of TWEAK-enhanced resorption rates, but surprisingly so did IL-27 neutralization. CONCLUSION: TWEAK is likely offering protection against the deleterious effects of TNF in implantation explaining embryo survival in a TNF-rich environment, and equal number of implants in both strains. However, there is a clear difference of protection in abortion-prone mating peaking in the abortion/resorption window but starting early, and therefore possible links with the prevention of abortion in CBA x DBA/2 matings by interfering with complement activation as recently described by Girardi et al. are discussed, as well as consequences for our current view of feto-maternal 'seed and soil' interplay. The apparently paradoxical effects of IL-27 neutralization are also discussed.

Endometrial vascularity by three-dimensional power Doppler ultrasound and cytokines: a complementary approach to assess uterine receptivity.

INTRODUCTION: Uterine receptivity was assessed simultaneously by measurement of vasoactive cytokines possibly involved in development of spiral arteries and by assessment of endometrial and uterine arterial blood flow. The objective was to explore the relationship between cytokine-related dysregulation and endometrial vascularisation in women with repeated implantation failures after in vitro fertilisation embryo transfer (IVF-ET). MATERIALS AND METHODS: We studied 40 women with recurrent IVF/ICSI-ET failures, despite replacement of more than 10 embryos of 'good quality', and 8 fertile controls. Three-dimensional ultrasound with power angiography was performed to record the sub-endometrial vascular flow index (VFI) and uterine artery pulsatility index prior to endometrial biopsy at day 21-23 of a monitored natural cycle. Endometrial IL-18, IL-18BP and IL-15 mRNA expression was assessed by real-time PCR, and the number of CD56(+) cells determined by immunochemistry. RESULTS: IL-18 and IL-15 mRNA expression was significantly different between the two groups. The range of variation in vascular imaging data was increased in the implantation failure (IF) group. The mRNA ratio for IL-15, but not the other cytokines, correlated with sub-endometrial vascular flow (r=0.65; p<0.001). This ratio correlated also with the mean number of CD56(+) cells per high-power field (r=0.41; p=0.005). Both IL-18 and IL-18BP mRNA expression was significantly negatively correlated with mean uterine artery pulsatility index (r=-0.37 and -0.43; p=0.02 and 0.01, respectively). CONCLUSION: Comprehensive ultrasonographic indicators appear to be related to various mechanisms, including insufficient or excessive uterine NK cell recruitment and inadequate endothelial vascular remodelling. New molecular tools may be useful in providing greater precision of uterine receptivity than ultrasonic indicators alone.

Cytokines: Important for implantation?

PROBLEM: Cytokines are obviously very important in an established pregnancy, but what about human embryo implantation? METHODS: Literature review. RESULTS: We first discuss the necessity and limits of animal models, and then review the few cytokines which have been demonstrated by knock-out methods to be absolutely necessary for embryo implantation using in animal models. We then review what is known or discussed about the role of other cytokines as deduced from quantitative and/or qualitative dysregulation in animals and in humans. CONCLUSIONS: Cytokines are indeed involved in implantation as they are in ongoing pregnancy and delivery. Relevance to infertility and recurrent pregnancy loss is discussed.

Uterine receptivity and cytokines: new concepts and new applications.

The implantation process, currently thought to be the most critical step in achieving a successful early pregnancy, remains one of the most important unsolved processes in reproductive medicine. It depends on uterine-dependent and embryo-specific events, which need to be critically coordinated. Early embryo signaling following a maternal hormonal or cytokine-mediated preparation phase seems to be involved in stages immediately before, during and just after the apposition step to permit adequate proliferation of the stroma. Our objective is to develop guidelines and diagnostic tools pertinent to appreciate uterine receptivity. We will focus our attention on the uterine luminal environment at the time of oocyte retrieval and on the monitoring of the endometrium using three-dimensional ultrasound associated with digital technology and cytokine quantification by real-time PCR during the implantation window in an IVF/ICSI population. There is an accumulating body of data which strongly suggests that both implantation and uterine receptivity are controlled, primarily, though not exclusively, by locally acting growth factors and cytokines, some under steroid control. Some specific cytokines (IL-12, IL-15 and IL-18) in the luminal environment and in the endometrium allow a distinct pattern of abnormal uterine receptivity. The identification of these distinct patterns of abnormal uterine receptivity and of the mechanisms leading to the abnormal angiogenesis before implantation strongly suggest that no single therapeutic scheme can correct all cases of implantation failure and should be adapted for each patient especially in the case of unexplained infertility.

Immune cells in uteroplacental tissues throughout pregnancy: a brief review.

In a brief introduction, this review states why the presence of immune cells at the interface poses problems for an immunologist (Medawar paradigm). Different types of placentation are then discussed, and the various interactions with leukocytes, the extreme being with the equids where a certain degree of 'attack' is often seen. The limits of animal models when dealing with the human situation are emphasized. It is then stated why the various phases of pregnancy are different, and an analysis made of the cellular movements at the implantation, peri-implantation, immediate post-implantation and resorption windows in rodents. Details of the cellular components involved are given, as are hints for the human situation. The Th1/Th2 paradigm is described, with clinical examples, and its limits. Thus, the newly appraised dual role of natural killer (NK) cells is discussed, with examples in rodents and in humans (pre-eclampsia, implantation failure, abortion systems). Clinical data on the IL-12/IL-18/NK tripod and implantation failure in humans are detailed.

Immune cells in uteroplacental tissues throughout pregnancy: a brief review.

In a brief introduction, this review states why the presence of immune cells at the interface poses problems for an immunologist (Medawar paradigm). Different types of placentation are then discussed, and the various interactions with leukocytes, the extreme being with the equids where a certain degree of 'attack' is often seen. The limits of animal models when dealing with the human situation are emphasized. It is then stated why the various phases of pregnancy are different, and an analysis made of the cellular movements at the implantation, peri-implantation, immediate post-implantation and resorption windows in rodents. Details of the cellular components involved are given, as are hints for the human situation. The Th1/Th2 paradigm is described, with clinical examples, and its limits. Thus, the newly appraised dual role of natural killer (NK) cells is discussed, with examples in rodents and in humans (pre-eclampsia, implantation failure, abortion systems). Clinical data on the IL-12 /IL-18/NK tripod and implantation failure in humans are detailed.

Downregulation of human endometrial IL-18 by exogenous ovarian steroids.

PROBLEM: To evaluate the influence of ovarian steroids on IL-18, IL-15 and angiopoietin-2 mRNA expression in the endometrium in the mid luteal phase. METHOD OF STUDY: We quantified IL-18/GAPDH, IL-18 BP/GAPDH, IL-15/GAPDH and angiopoietin-2/GAPDH in the endometrium by quantitative polymerase chain reaction on day 21 of the cycle. We first compared cytokines expression over two natural cycles (n = 15) then between natural and oestrogen-progestin replacement treatment (n = 18). RESULTS: Endometrial IL-18, IL-18 BP, IL-15 and angiopoietin-2 mRNA expression did not change over two natural cycles. Addition of exogenous hormones significantly decreased IL-18 and IL-18 BP mRNA expression but not influence IL-15 or angiopoietin-2 ratios. This was also observed with immunohistochemistry. CONCLUSION: Exogenous oestro-progestative hormones influence endometrial IL-18 system expression involved in angiogenesis and in the uterine natural killer (uNK) cell activation pathway during the implantation process.

Cytokines and implantation.

This review introduces the field of cytokines and implantation and then recalls the specialized role of the uterus and the notion of the 'implantation window'. The role of inflammatory and angiogenic cytokines is presented, as well as the involvement of cytokines such as -interferons in corpus luteum maintenance in non-chorionic gonadotrophin-producing species. -Interferons are reviewed, before dealing with the more in depth analysis of cytokine networks in the pre- and peri-implantation uterus. The emerging involvement of cytokines in controlling uterine vascularization angiogenesis is reviewed, with emphasis on NK activating factors.

Immunological similarities between implantation and pre-eclampsia.

PROBLEM: Cytokines are involved in implantation success and failure. We envisage that they could be similarly involved in pre-eclampsia (PE). MATERIALS AND METHODS: First, we review the primipaternity and primiparity concepts and then why natural killer (NK) cells are involved in implantation. We stress that the common event in all PE is vascular remodelling. RESULTS AND CONCLUSION: We conclude that PE could involve cytokine and/or NK dysfunctions, and propose a working hypothesis.

Role of the endometrial tripod interleukin-18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization-embryo transfer failure.

OBJECTIVE: To document in the endometrium the correlation among the interleukin (IL)-12, -15, and -18 mRNA and the correlation between cytokine levels, vascular status, and endometrial natural killer (NK) cell count in the context of recurrent implantation failure. DESIGN: A pilot study. SETTING: Department of Reproductive Immunology. PATIENT(S): Women who failed to become pregnant after repeated IVF-embryo transfer and fertile control subjects. INTERVENTION(S): Ultrasonic evaluation and endometrial biopsy in luteal phase. MAIN OUTCOME MEASURE(S): Uterine artery Doppler, count of uterine CD56 bright cells/field, and quantification by real-time polymerase chain reaction (PCR) to monitor IL-12 family (IL-12p40, IL-12p35, EBI3, IL-23), the IL-18 system (IL-18, IL-18R, IL18BP), and the IL-15 mRNA ratio. RESULT(S): The uterine artery Doppler and the CD56 bright cell counts were significantly different in fertile and infertile patients. The mean uterine artery pulsatility index correlated significantly negatively with the IL-18/actin ratio suggesting a defect of the cytokine-dependent vascular remodeling pathway. The number of uterine CD56 bright cells was significantly correlated with the IL-15/actin and IL-18/IL-18BP ratios. Thus, IL-18 and IL-15 seems to be involved in the local recruitment and the activation of uterine natural killer (uNK) cells. IL-18 was itself correlated with IL-15 and IL-12, suggesting a local control of uNK cells activation. CONCLUSION(S): The assessment of the tripod IL-12/-15/-18 shows distinct immune-related mechanisms that are involved in the broader context of inadequate uterine receptivity.