NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium are precursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

A combination of immune cell types identified through ensemble machine learning strategy detects altered profile in recurrent pregnancy loss: a pilot study.

OBJECTIVE: To compare the immunologic profiles of peripheral and menstrual blood (MB) of women who experience recurrent pregnancy loss and women without pregnancy complications. DESIGN: Explorative case-control study. Cross-sectional assessment of flow cytometry-derived immunologic profiles. SETTING: Academic medical center. PATIENT(S): Women who experienced more than 2 consecutive miscarriages. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Flow cytometry-based immune profiles of uterine and systemic immunity (recurrent pregnancy loss, n = 18; control, n = 14) assessed by machine learning classifiers in an ensemble strategy, followed by recursive feature selection. RESULT(S): In peripheral blood, the combination of 4 cell types (nonswitched memory B cells, CD8+ T cells, CD56bright CD16- natural killer [NKbright] cells, and CD4+ effector T cells) classified samples correctly to their respective cohort. The identified classifying cell types in peripheral blood differed from the results observed in MB, where a combination of 6 cell types (Ki67+CD8+ T cells, (Human leukocyte antigen-DR+) regulatory T cells, CD27+ B cells, NKbright cells, regulatory T cells, and CD24HiCD38Hi B cells) plus age allowed for assigning samples correctly to their respective cohort. Based on the combination of these features, the average area under the curve of a receiver operating characteristic curve and the associated accuracy were >0.8 for both sample sources. CONCLUSION(S): A combination of immune subsets for cohort classification allows for robust identification of immune parameters with possible diagnostic value. The noninvasive source of MB holds several opportunities to assess and monitor reproductive health.

Antibiotic Intervention Affects Maternal Immunity During Gestation in Mice.

Background: Pregnancy is a portentous stage in life, during which countless events are precisely orchestrated to ensure a healthy offspring. Maternal microbial communities are thought to have a profound impact on development. Although antibiotic drugs may interfere in these processes, they constitute the most frequently prescribed medication during pregnancy to prohibit detrimental consequences of infections. Gestational antibiotic intervention is linked to preeclampsia and negative effects on neonatal immunity. Even though perturbations in the immune system of the mother can affect reproductive health, the impact of microbial manipulation on maternal immunity is still unknown. Aim: To assess whether antibiotic treatment influences maternal immunity during pregnancy. Methods: Pregnant mice were treated with broad-spectrum antibiotics. The maternal gut microbiome was assessed. Numerous immune parameters throughout the maternal body, including placenta and amniotic fluid were investigated and a novel machine-learning ensemble strategy was used to identify immunological parameters that allow distinction between the control and antibiotic-treated group. Results: Antibiotic treatment reduced diversity of maternal microbiota, but litter sizes remained unaffected. Effects of antibiotic treatment on immunity reached as far as the placenta. Four immunological features were identified by recursive feature selection to contribute to the most robust classification (splenic T helper 17 cells and CD5+ B cells, CD4+ T cells in mesenteric lymph nodes and RORγT mRNA expression in placenta). Conclusion: In the present study, antibiotic treatment was able to affect the carefully coordinated immunity during pregnancy. These findings highlight the importance of inclusion of immunological parameters when studying the effects of medication used during gestation.

Clusters of Tolerogenic B Cells Feature in the Dynamic Immunological Landscape of the Pregnant Uterus.

Well-timed interaction of correctly functioning maternal immune cells is essential to facilitate healthy placenta formation, because the uterine immune environment has to tolerate the semi-allogeneic fetus and allow adequate trophoblast invasion. Here, we assess the uterine immune signature before and during pregnancy. Extensive supervised and unsupervised flow cytometry clustering strategies not only show a general increase in immune memory throughout pregnancy but also reveal the continuous presence of B cells. Contrary to the belief that B cells are merely a consequence of uterine pathology, decidual B cells produce IL-10 and are found to be localized in clusters, together with Foxp3pos T cells. Our findings therefore suggest a role for B cells in healthy pregnancy.

Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface.

During pregnancy, maternal regulatory T cells (Tregs) are important in establishing immune tolerance to invading fetal extravillous trophoblasts (EVTs). CD25HIFOXP3+ Tregs are found at high levels in decidual tissues and have been shown to suppress fetus-specific and nonspecific responses. However, limited data are available on additional decidual Treg types and the mechanisms by which they are induced. This study investigated three distinct decidual CD4+ Treg types in healthy pregnancies with a regulatory phenotype and the ability to suppress T cell responses: CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim. Moreover, co-culture of HLA-G+ EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportions of CD25HIFOXP3+ Tregs compared to T cells cultured alone. EVTs also increased PD1HI Tregs that could be inhibited by HLA-C and CD3 antibodies, suggesting an antigen-specific induction. The presence of distinct Treg types may allow for the modulation of a variety of inflammatory responses in the placenta.

How uterine microbiota might be responsible for a receptive, fertile endometrium.

BACKGROUND: Fertility depends on a receptive state of the endometrium, influenced by hormonal and anatomical adaptations, as well as the immune system. Local and systemic immunity is greatly influenced by microbiota. Recent discoveries of 16S rRNA in the endometrium and the ability to detect low-biomass microbiota fueled the notion that the uterus may be indeed a non-sterile compartment. To date, the concept of the 'sterile womb' focuses on in utero effects of microbiota on offspring and neonatal immunity. However, little awareness has been raised regarding the importance of uterine microbiota for endometrial physiology in reproductive health; manifested in fertility and placentation. OBJECTIVE AND RATIONALE: Commensal colonization of the uterus has been widely discussed in the literature. The objective of this review is to outline the possible importance of this uterine colonization for a healthy, fertile uterus. We present the available evidence regarding uterine microbiota, focusing on recent findings based on 16S rRNA, and depict the possible importance of uterine colonization for a receptive endometrium. We highlight a possible role of uterine microbiota for host immunity and tissue adaptation, as well as conferring protection against pathogens. Based on knowledge of the interaction of the mucosal immune cells of the gut with the local microbiome, we want to investigate the potential implications of commensal colonization for uterine health. SEARCH METHODS: PubMed and Google Scholar were searched for articles in English indexed from 1 January 2008 to 1 March 2018 for '16S rRNA', 'uterus' and related search terms to assess available evidence on uterine microbiome analysis. A manual search of the references within the resulting articles was performed. To investigate possible functional contributions of uterine microbiota to health, studies on microbiota of other body sites were additionally assessed. OUTCOMES: Challenging the view of a sterile uterus is in its infancy and, to date, no conclusions on a 'core uterine microbiome' can be drawn. Nevertheless, evidence for certain microbiota and/or associated compounds in the uterus accumulates. The presence of microbiota or their constituent molecules, such as polysaccharide A of the Bacteroides fragilis capsule, go together with healthy physiological function. Lessons learned from the gut microbiome suggest that the microbiota of the uterus may potentially modulate immune cell subsets needed for implantation and have implications for tissue morphology. Microbiota can also be crucial in protection against uterine infections by defending their niche and competing with pathogens. Our review highlights the need for well-designed studies on a 'baseline' microbial state of the uterus representing the optimal starting point for implantation and subsequent placenta formation. WIDER IMPLICATIONS: The complex interplay of processes and cells involved in healthy pregnancy is still poorly understood. The correct receptive endometrial state, including the local immune environment, is crucial not only for fertility but also placenta formation since initiation of placentation highly depends on interaction with immune cells. Implantation failure, recurrent pregnancy loss, and other pathologies of endometrium and placenta, such as pre-eclampsia, represent an increasing societal burden. More robust studies are needed to investigate uterine colonization. Based on current data, future research needs to include the uterine microbiome as a relevant factor in order to understand the players needed for healthy pregnancy.

Human uterine lymphocytes acquire a more experienced and tolerogenic phenotype during pregnancy.

Pregnancy requires a delicate immune balance that nurtures the allogeneic fetus, while maintaining reactivity against pathogens. Despite increasing knowledge, data is lacking on the transition of pre-pregnancy endometrial lymphocytes to a pregnancy state. Here, we immunophenotyped lymphocytes from endometrium (MMC), term decidua parietalis (DPMC), and PBMC for direct comparison. We found that the immune cell composition of MMC and DPMC clearly differ from each other, with less NK-cells, and more NKT-cells and T-cells in DPMC. An increased percentage of central memory and effector memory T-cells, and less naive T-cells in DPMC indicates that decidual T-cells are more experienced than endometrial T-cells. The increased percentage of CD4+CD25highCD127- Treg in DPMC, including differentiated Treg, is indicative of a more experienced and tolerogenic environment during pregnancy. The Th cell composition of both MMC and DPMC was different from PBMC, with a preference for Th1 over Th2 in the uterine environment. Between MMC and DPMC, percentages of Th cell subsets did not differ significantly. Our results suggest that already before pregnancy a tightly controlled Th1/Th2/Th17 balance is present. These findings create opportunities to further investigate the underlying immune mechanism of pregnancy complications using menstrual blood as a source for endometrial lymphocytes.

Heparanase Is Essential for the Development of Acute Experimental Glomerulonephritis.

Heparanase, a heparan sulfate (HS)--specific endoglucuronidase, mediates the onset of proteinuria and renal damage during experimental diabetic nephropathy. Glomerular heparanase expression is increased in most proteinuric diseases. Herein, we evaluated the role of heparanase in two models of experimental glomerulonephritis, being anti-glomerular basement membrane and lipopolysaccharide-induced glomerulonephritis, in wild-type and heparanase-deficient mice. Induction of experimental glomerulonephritis led to an increased heparanase expression in wild-type mice, which was associated with a decreased glomerular expression of a highly sulfated HS domain, and albuminuria. Albuminuria was reduced in the heparanase-deficient mice in both models of experimental glomerulonephritis, which was accompanied by a better renal function and less renal damage. Notably, glomerular HS expression was preserved in the heparanase-deficient mice. Glomerular leukocyte and macrophage influx was reduced in the heparanase-deficient mice, which was accompanied by a reduced expression of both types 1 and 2 helper T-cell cytokines. In vitro, tumor necrosis factor-α and lipopolysaccharide directly induced heparanase expression and increased transendothelial albumin passage. Our study shows that heparanase contributes to proteinuria and renal damage in experimental glomerulonephritis by decreasing glomerular HS expression, enhancing renal leukocyte and macrophage influx, and affecting the local cytokine milieu.