Fiona M. Menzies

Sequential expression of macrophage anti-microbial/inflammatory and wound healing markers following innate, alternative and classical activation

The present study examines the temporal dynamics of macrophage activation marker expression in response to variations in stimulation. We demonstrate that markers can be categorized as ‘early’ (expressed most abundantly at 6 h post‐stimulation) or ‘late’ (expressed at 24 h post‐stimulation). Thus nos2 and p40 (IL‐12/IL‐23) are early markers of innate and classical activation, while dectin‐1 and mrc‐1 are early markers and fizz1 (found in inflammatory zone‐1) and ym1 are late markers of alternative activation. Furthermore, argI is a late marker of both innate and alternative activation. The ability of interferon (IFN)‐γ to alter these activation markers was studied at both the protein level and gene level. As reported previously, IFN‐γ was able to drive macrophages towards the classical phenotype by enhancing nos2 gene expression and enzyme activity and p40 (IL‐12/IL‐23) gene expression in lipopolysaccharide (LPS)‐stimulated macrophages. IFN‐γ antagonized alternative macrophage activation, as evident by reduced expression of dectin‐1, mrc‐1, fizz1 and ym1 mRNA transcripts. In addition, IFN‐γ antagonized arginase activity irrespective of whether macrophages were activated innately or alternatively. Our data explain some apparent contradictions in the literature, demonstrate temporal plasticity in macrophage activation states and define for the first time ‘early’ and ‘late’ markers associated with anti‐microbial/inflammatory and wound healing responses, respectively.

Chemokine scavenger D6 is expressed by trophoblasts and aids the survival of mouse embryos transferred into allogeneic recipients.

Proinflammatory CC chemokines are thought to drive recruitment of maternal leukocytes into gestational tissues and regulate extravillous trophoblast migration. The atypical chemokine receptor D6 binds many of these chemokines and is highly expressed by the human placenta. D6 is thought to act as a chemokine scavenger because, when ectopically expressed in cell lines in vitro, it efficiently internalizes proinflammatory CC chemokines and targets them for destruction in the absence of detectable chemokine-induced signaling. Moreover, D6 suppresses inflammation in many mouse tissues, and notably, D6-deficient fetuses in D6-deficient female mice show increased susceptibility to inflammation-driven resorption. In this paper, we report strong anti-D6 immunoreactivity, with specific intracellular distribution patterns, in trophoblast-derived cells in human placenta, decidua, and gestational membranes throughout pregnancy and in trophoblast disease states of hydatidiform mole and choriocarcinoma. We show, for the first time, that endogenous D6 in a human choriocarcinoma-derived cell line can mediate progressive chemokine scavenging and that the D6 ligand CCL2 can specifically associate with human syncytiotrophoblasts in term placenta in situ. Moreover, despite strong chemokine production by gestational tissues, levels of D6-binding chemokines in maternal plasma decrease during pregnancy, even in women with pre-eclampsia, a disease associated with increased maternal inflammation. In mice, D6 is not required for syngeneic or semiallogeneic fetal survival in unchallenged mice, but interestingly, it does suppress fetal resorption after embryo transfer into fully allogeneic recipients. These data support the view that trophoblast D6 scavenges maternal chemokines at the fetomaternal interface and that, in some circumstances, this can help to ensure fetal survival.

The role of mast cells and their mediators in reproduction, pregnancy and labour

BACKGROUND Mast cells (MCs) are the classical mediators of allergy, however, their importance in the development of innate and adaptive immune responses is increasingly being recognized. Herein, the present MC literature is summarized, with particular focus on studies of MCs in the endometrium and myometrium, and their involvement in fertility, implantation, pregnancy and labour. METHODS Recent developments in MC biology were identified by systematic searches of PubMed, Medline and Google Scholar from 2000 to November 2009. To specifically examine the role of MCs in fertility and pregnancy, we then performed a systematic review of English literature cited in the PubMed, Medline and Google Scholar databases, but extended the search period, from 1980 to January 2010 RESULTS MCs can respond to immunoglobulin E-independent innate immune stimuli and are present within the endometrium, with activation and release of mediators occurring prior to menstruation and in association with endometriosis. With respect to pregnancy, MCs are redundant during blastocyst implantation and although their mediators can induce myometrial contractility, there is no epidemiological link of preterm birth with allergy, suggesting a non-essential role or robust regulation. In males, MCs are present in the testes and are increased in oligo- and azoospermia, with MC mediators directly suppressing sperm motility in a potentially reversible manner. CONCLUSIONS MCs are prevalent in the female and male reproductive tract. However, whether MCs are absolutely required for a successful pregnancy or are fundamental to reproductive pathology, and thereby a therapeutic target, remains to be determined.

Selective inhibition and augmentation of alternative macrophage activation by progesterone

Progesterone is the female sex hormone necessary for the maintenance of pregnancy, and is known to modulate macrophage activation. However, studies have concentrated exclusively on the ability of progesterone to negatively regulate the innate and classical pathways of activation, associated with nitric oxide (NO) and interleukin (IL)‐12 production. Our aim was to examine the ability of progesterone to modulate alternative macrophage activation. Bone marrow cells were isolated and differentiated from male BALB/c mice, exposed to varying concentrations of progesterone and stimulated with lipopolysaccharide (LPS) (innate activation), IL‐4 (alternative activation) or LPS in combination with IL‐4. Our present study demonstrates that progesterone not only down‐regulates inducible nitric oxide synthase 2 (iNOS) activity in macrophages but also arginase activity, in a dose‐dependent manner, independent of the stimuli, whether it is induced by LPS (innate activation), IL‐4 (alternative activation) or LPS in combination with IL‐4. The ability of progesterone to down‐modulate IL‐4‐induced cell surface expression of the mannose receptor further suggested a negative regulation of alternative macrophage activation by this hormone. Analysis of mRNA expression, by quantitative reverse transcription–polymerase chain reaction (qRT–PCR), of genes associated with innate and alternative macrophage activation revealed that progesterone down‐regulated LPS‐induced macrophage nos2, argI and p40 (IL‐12/IL‐23) expression and IL‐4‐induced argI, mrc‐1 and fizz1 expression. However, progesterone up‐regulated IL‐4‐induced macrophage expression of ym1, while dectin‐1 expression remained unaltered. Following treatment of macrophages with LPS and IL‐4 in combination a similar pattern was observed, with the exception that progesterone up‐regulated macrophage expression of fizz1 as well as ym1 and did not modify mrc‐1 expression. Our data demonstrate for the first time that a hormone has the ability to regulate selectively the expression of different genes associated with alternative macrophage activation.

Mast cells reside in myometrium and cervix, but are dispensable in mice for successful pregnancy and labor

Parturition is associated with myometrial and cervical inflammation. The causes and consequences of this inflammatory response are not clear. Mast cells (MCs) are important inducers of allergic and non‐allergic inflammation, and their secreted products can induce myometrial contractions. Thus, mast cell activation has been hypothesized to have a role in initiating labor and/or driving labor‐associated inflammation. We report that small numbers of MCs expressing chymase and tryptase are present in the myometrium and cervix of pregnant women. Labor did not lead to any change in mast cell abundance in these tissues, but was associated with reduced expression of the mast‐cell regulator FcεR1A, indicative of a change in mast cell properties. This coincided with contraction‐dependent myocyte production of interleukin‐10 (IL‐10), a known suppressor of FcεR1A expression. MCs were also found in the uterine horn and cervical region of pregnant C57BL/6 mice, increasing in number in the cervix, but not the myometrium, with labor. As expected, these cells were absent from mast‐cell‐deficient KitW−sh mice. Nonetheless, pregnant KitW−sh mice showed no defects in the timing of labor induction or in the upregulation of leukocyte markers during labor. Thus, MCs are present in the uterus and cervix of humans and mice, and our mouse studies suggest that they do not have a vital role in the induction of labor, or in the promotion of labor‐associated inflammation.

The Chemokine Receptor CCR2 Is Not Required for Successful Initiation of Labor in Mice

ABSTRACT Chemokine-driven neutrophil and monocyte recruitment into the uterus and cervix has been proposed to initiate labor. Chemokines that bind CXCR2 direct neutrophil migration and are induced during labor in humans. The chemokine CCL2, induced in the uterus by endocrine and mechanical signals, has been proposed to drive CCR2-dependent monocyte homing to the uterus to contribute to the initiation of labor. However, no direct evidence indicates that chemokines or their receptors play indispensable roles in labor-associated inflammation, and the impact of leukocyte infiltration on labor is unclear. Here, we have quantified expression of the principal monocyte- and neutrophil-attracting chemokines in the uteri of term pregnant (Day 18) and laboring wild-type mice. None of the neutrophil attractants we assayed were up-regulated with labor. Strikingly, however, Ccl2 was markedly increased, and this was concomitant with increased expression of Ccr2, the myeloid marker Itgam (also known as Cd11b), the monocyte/macrophage marker Emr1 (also known as F4/80). Moreover, in CCR2-deficient mice, this labor-associated increase in Itgam and Emr1 was not seen, consistent with the monocyte-trafficking defects that exist in these animals. Nonetheless, laboring CCR2-deficient and wild-type uteri showed similarly enhanced expression of the myometrial activation markers Gja1 and Oxtr (commonly known as connexin 43 and oxytocin receptor, respectively), and CCR2-deficient mice had gestation lengths, litter sizes, and fetal and placental weights no different from those of their wild-type counterparts. Thus, whereas labor is associated with an inflammatory response in gestational tissues, CCR2-dependent leukocyte recruitment into the mouse uterus is dispensable for the initiation of successful labor.

Immunomodulation by the Female Sex Hormones

Pregnancy is a highly regulated process, requiring strict control of the immune system in order to prevent rejection of the semiallogenic foetus. One aspect of pregnancy immunology that has been of great interest is the influence of female sex and pregnancy associated hormones, such as progesterone and oestrogen, on cells of the immune system. This review evaluates studies investigating the ability of these hormones to modulate the function of cells of both the innate and adaptive arms of the immune system and mechanisms by which immunity to infection can be altered due to increased levels of progesterone and oestrogen. Finally, the influence of pregnancy on the most common autoimmune diseases, on toxoplasmosis and on malaria is reviewed. Pregnancy is a complex process in which cells and molecules of the maternal immune system interact in such a way as to prevent the rejection of the semiallogenic foetus. Development and modulation of pregnancy is controlled by the presence and levels of various sex and pregnancy associated hormones, such as oestrogens and progesterones. In addition, the cytokine environment is important for a successful pregnancy, with studies showing a Th1 environment to be associated with abortion and a Th2 environment allowing the successful continuation of pregnancy (1). The elevated levels of oestrogen and progesterone observed during pregnancy possess a number of modulatory functions on cells of the immune system, including macrophages, natural killer (NK) cells, dendritic cells (DCs), T cells and B cells. The endometrium is one of the most important organs during pregnancy (2) and presents an immunologically competent environment, with 30% of cells being of the immune system (3). Within the uterus of non- pregnant women, NK cells, macrophages, T cells and B cells are abundant (2-4) and inhibit the implantation and the development of the embryo. However, during pregnancy, the activity of these cells against the foetus is specifically suppressed by oestrogen and progesterone, allowing successful embryo implantation (5-9).

Atypical Chemokine Receptor ACKR2 Mediates Chemokine Scavenging by Primary Human Trophoblasts and Can Regulate Fetal Growth, Placental Structure, and Neonatal Mortality in Mice

Inflammatory chemokines produced in the placenta can direct the migration of placental leukocytes using chemokine receptors that decorate the surface of these cells. Fetal trophoblasts can also express receptors for inflammatory chemokines, and they are one of the few cell types that express atypical chemokine receptor 2 (ACKR2), previously known as D6. ACKR2 binds many inflammatory CC chemokines but cannot stimulate cell migration or activate signaling pathways used by conventional chemokine receptors. Existing evidence suggests that ACKR2 is a specialized chemokine scavenger, but its function in primary human trophoblasts has not been explored. In mice, ACKR2 is thought to be dispensable for the reproductive success of unchallenged females that have conceived naturally, but it can suppress inflammation-induced abortion and aid the survival of implanted allogeneic embryos. In this article, we demonstrate that cultured primary human trophoblasts express ACKR2 far more strongly than genes encoding conventional receptors for inflammatory CC chemokines. Moreover, these cells are capable of the rapid internalization and efficient scavenging of extracellular chemokine, and this is mediated by ACKR2. We also report that in unchallenged DBA/1j mice, Ackr2 deficiency increases the incidence of stillbirth and neonatal death, leads to structural defects in the placenta, and can decrease fetal weight. Loss of Ackr2 specifically from fetal cells makes a key contribution to the placental defects. Thus, primary human trophoblasts use ACKR2 to scavenge chemokines, and ACKR2 deficiency can cause abnormal placental structure and reduced neonatal survival.

Pregnancy and Susceptibility to Parasites

Successful pregnancy is dependent on many dynamic immunological events that occur at the maternal–fetal interface. While the trophoblast initiates implantation through the uterine wall, maternal immune mechanisms limit the extent to which the decidua is invaded by the placenta without disrupting pregnancy. Immunological reactions are not observed at the maternal–fetal interface. Pregnancy-induced modulation of systemic maternal immunity results in amelioration of certain autoimmune diseases and exacerbation of some infectious diseases. Alteration of immunological function during pregnancy in some circumstances might facilitate congenital transmission of certain pathogens. In other circumstances, the immune reactions induced by pathogens might result in disruption of pregnancy. These complex interactions are discussed in the context of a number of diseases caused by infection with taxonomically diverse protozoan parasites, nematodes, and cestodes.

The role of chemokines and their receptors during protist parasite infections.

Protists are a diverse collection of eukaryotic organisms that account for a significant global infection burden. Often, the immune responses mounted against these parasites cause excessive inflammation and therefore pathology in the host. Elucidating the mechanisms of both protective and harmful immune responses is complex, and often relies of the use of animal models. In any immune response, leucocyte trafficking to the site of infection, or inflammation, is paramount, and this involves the production of chemokines, small chemotactic cytokines of approximately 8-10 kDa in size, which bind to specific chemokine receptors to induce leucocyte movement. Herein, the scientific literature investigating the role of chemokines in the propagation of immune responses against key protist infections will be reviewed, focussing on Plasmodium species, Toxoplasma gondii, Leishmania species and Cryptosporidium species. Interestingly, many studies find that chemokines can in fact, promote parasite survival in the host, by drawing in leucocytes for spread and further replication. Recent developments in drug targeting against chemokine receptors highlights the need for further understanding of the role played by these proteins and their receptors in many different diseases.