Macrophages and small extracellular vesicle mediated-intracellular communication in the peritoneal microenvironment: impact on endometriosis development

Endometriosis is an inflammatory disease that is defined as the growth of endometrium-like tissue outside the uterus, commonly on the lining of the pelvic cavity, visceral organs and in the ovaries. It affects around 190 million women of reproductive age worldwide and is associated with chronic pelvic pain and infertility, which greatly impairs health-related life quality. The symptoms of the disease are variable, this combined with a lack of diagnostic biomarkers and necessity of surgical visualisation to confirm disease, the prognosis can take an average timespan of 6–8 years. Accurate non-invasive diagnostic tests and the identification of effective therapeutic targets are essential for disease management. To achieve this, one of the priorities is to define the underlying pathophysiological mechanisms that contribute to endometriosis. Recently, immune dysregulation in the peritoneal cavity has been linked to endometriosis progression. Macrophages account for over 50% of immune cells in the peritoneal fluid and are critical for lesion growth, angiogenesis, innervation and immune regulation. Apart from the secretion of soluble factors like cytokines and chemokines, macrophages can communicate with other cells and prime disease microenvironments, such as the tumour microenvironment, via the secretion of small extracellular vesicles (sEVs). The sEV-mediated intracellular communication pathways between macrophages and other cells within the peritoneal microenvironment in endometriosis remain unclear. Here, we give an overview of peritoneal macrophage (pMΦ) phenotypes in endometriosis and discuss the role of sEVs in the intracellular communication within disease microenvironments and the impact they may have on endometriosis progression

The remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

The αβ T-cell co-receptor CD4 enhances immune responses more than one million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native co-receptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in two dimensions (2D) using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D dissociation constant, Kd, of ~5000 molecules/μm2. This value is 2-3 orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by three-fold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore appears to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.This work was supported by the Wellcome Trust and the UK Medical Research Council. PJ was supported by grants from the Swedish Research Council (number: 623-2014- 6387 and 621-2014-3907). OD is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 098363)

ST2 and IL-33 in Pregnancy and Pre-Eclampsia

Normal pregnancy is associated with a mild systemic inflammatory response and an immune bias towards type 2 cytokine production, whereas pre-eclampsia is characterized by a more intense inflammatory response, associated with endothelial dysfunction and a type 1 cytokine dominance. Interleukin (IL)-33 is a newly described member of the IL-1 family, which binds its receptor ST2L to induce type 2 cytokines. A soluble variant of ST2 (sST2) acts as a decoy receptor to regulate the activity of IL-33. In this study circulating IL-33 and sST2 were measured in each trimester of normal pregnancy and in women with pre-eclampsia. While IL-33 did not change throughout normal pregnancy, or between non-pregnant, normal pregnant or pre-eclamptic women, sST2 was significantly altered. sST2 was increased in the third trimester of normal pregnancy (p<0.001) and was further increased in pre-eclampsia (p<0.001). This increase was seen prior to the onset of disease (p<0.01). Pre-eclampsia is a disease caused by placental derived factors, and we show that IL-33 and ST2 can be detected in lysates from both normal and pre-eclampsia placentas. ST2, but not IL-33, was identified on the syncytiotrophoblast layer, whereas IL-33 was expressed on perivascular tissue. In an in vitro placental perfusion model, sST2 was secreted by the placenta into the ‘maternal’ eluate, and placental explants treated with pro-inflammatory cytokines or subjected to hypoxia/reperfusion injury release more sST2, suggesting the origin of at least some of the increased amounts of circulating sST2 in pre-eclamptic women is the placenta. These results suggest that sST2 may play a significant role in pregnancies complicated by pre-eclampsia and increased sST2 could contribute to the type 1 bias seen in this disorder

Multicolor Flow Cytometry and Nanoparticle Tracking Analysis of Extracellular Vesicles in the Plasma of Normal Pregnant and Pre-eclamptic Women1

Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied

Measurement of sST2 is comparable to PlGF in the diagnosis of early-onset pre-eclampsia

A diagnostic test to confirm pre-eclampsia would be beneficial for the clinical management of the syndrome. The Triage PlGF test is able to confirm pre-eclampsia with high accuracy, with the greatest efficacy at <35 weeks gestation. We recently found that the anti-inflammatory protein sST2 is elevated in the plasma of pre-eclamptic women compared to normal controls. Here sST2 and PlGF are compared in early-onset and late-onset pre-eclamptic women. sST2 was found to be an equally good diagnostic tool for early-onset (sST2 AUC 0.944 versus PlGF AUC 0.995; not significant) but not late-onset disease. © 2013 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved

Circulating IL-33 in pregnancy and pre-eclampsia.

<p>A) IL-33 was detected by ELISA in the serum from women with a diagnosis of pre-eclampsia, matched controls who had a normal pregnancy and matched non-pregnant women (n = 20). No significant differences in IL-33 were identified. Bars represent median values. B) IL-33 was detected by ELISA in samples taken throughout pregnancy in women who developed pre-eclampsia and matched controls who had an uncomplicated pregnancy (n = 15). Blood samples were taken in each trimester of pregnancy (1, 2, 3) and at the diagnosis of pre-eclampsia (PE), and compared to non-pregnant matched women. There were no significant differences throughout pregnancy or between normal and pre-eclampsia pregnancies. Bars represent median values.</p

Clinical characteristics of women with pre-eclampsia, normal pregnant women and non-pregnant women (n = 20).

<p>Data are shown as mean (+/− standard deviation).</p

Circulating sST2 in pregnancy and pre-eclampsia.

<p>A) sST2 was detected by ELISA in the plasma from women with a diagnosis of pre-eclampsia, matched controls who had a normal pregnancy and matched non-pregnant women (n = 20). sST2 was significantly raised in pre-eclampsia compared to normal pregnancy (p<0.001). B) sST2 was detected by ELISA in samples taken throughout pregnancy in women who developed pre-eclampsia and matched controls who had an uncomplicated pregnancy (n = 15). Blood samples were taken in each trimester of pregnancy (1, 2, 3) and at the diagnosis of pre-eclampsia (PE), and compared to non-pregnant matched women. There is a significant increase in plasma sST2 in the third trimester of normal pregnancy compared to the first or second trimester (p<0.001), and compared to the non-pregnant group (p<0.001). sST2 was significantly raised in pre-eclampsia compared to normal pregnancy during the third trimester (p<0.01), prior to the onset of clinical symptoms. Levels further increased when pre-eclampsia symptoms presented (p<0.05). Bars represent mean values, * = p<0.05,** = p<0.01, *** = p<0.001. C) ROC curve analysis indicates sST2 levels in the third trimester sample (n = 10) are likely to predict pre-eclampsia (AUC = 0.813).</p

Expression of IL-33 and sST2 by the placenta.

<p>A) IL-33 was detected by western blotting of placental lysates from normal and pre-eclampsia placentas (n = 9). Both the full length (closed arrow) and cleaved forms (open arrow) were detected. ST2 was detected in each of the placental lysates. B) Densitometry of protein bands standardised to actin expression revealed no change in the levels of IL-33 nor sST2 between normal and pre-eclamptic placentas. C–H) Expression of IL-33 and sST2 can be seen in by immunocytochemical analysis of both normal (n = 3) and pre-eclampsia (n = 3) placentas (C-E and F-H show staining from two representative placentas respectively).</p