HEALTH ADVANTAGES

REDUCTION OF MULTIPLE SCLEROSIS ACTIVITY

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Mounting evidence suggests that the required immune regulation during pregnancy is not simply a general ‘immunosuppressive state’, but modulates the way the adaptive immune system, specifically T cells, respond to self and foreign antigens. Intriguing clues to the importance of this immune modulation come from clinical observations in autoimmune diseases including multiple sclerosis (MS). Several studies have shown that inflammatory disease activity and risk for MS relapses decreases throughout pregnancy, but re-bounds after delivery. The reduction of relapse rate in the third trimester is estimated to reach up to 80%, which is a far larger reduction in disease activity than that achieved by currently available MS therapies. Interestingly, in the MS animal model, experimental autoimmune encephalomyelitis (in short EAE), pregnant mice are also protected from disease but show exacerbated neuroinflammation after delivery.

The aim of this project is to understand the mechanisms responsible for pregnancy mediated immune regulation and its benefit on maternal autoimmunity. Based on our preliminary data, we hypothesize that the pathways mediating tolerance against fetal antigens during pregnancy also affect responses against self-antigens, thereby ameliorating autoimmune diseases. Driven by cell-mediated and/or hormonal regulation, this is presumed to result in a shift in the maternal T cell repertoire and temporary induction of antigen-specific immune tolerance.

To test this hypothesis, our translational project is subdivided into three main aims, each of which will be explored in the animal model EAE as well as a clinical prospective cohort of female MS patients over the course of pregnancy and postpartum.

Firstly, we will explore clonal composition of the T cell repertoire as well as characterize in detail self- and foreign-reactive T cell responses during pregnancy using a combination of next generation sequencing of the T cell repertoire, and functional probing of antigen-specific T cell responses. In parallel and with similar methods, we will assess the mouse T cell repertoire in pregnant, post-pregnant and virgin mice with EAE and healthy controls.

Secondly, we want to identify relevant major T cell subpopulations mediating pregnancy-associated advantages for maternal health. Based on the results of our first objective, we will study the most strongly regulated T cell subsets by performing in depth flow cytometric phenotyping. Again, this will be done similarly in human and mouse samples. Moreover the mouse model will allow us to examine the antigen specificity and the functionality of these T cell subsets in more detail.

Thirdly, we want to investigate the interaction of pregnancy-related hormones with self-reactive and foreign-reactive T cell responses. To assess the effect of pregnancy-related hormones on differentially regulated T cell subsets throughout pregnancy in both the mouse model and human samples, single cell analysis will be used to quantify the gene expression of T cell differentiation, T cell activation and steroid signaling. Finally, cell-specific steroid receptor knock-out mice will be used to compare frequency, phenotype and function of T cell subpopulations in pregnant and non-pregnant EAE animals.

This translational project will complement the projects "WHY DO WOMEN DEVELOP SEVERE INFLUENZA ILLNESS DURING PREGNANCY?" and "INFLUENCE OF PREGNANCY-RELATED HORMONES ON IMMUNITY" to provide a comprehensive picture of the “costs and benefits” of feto-maternal immune cross talk and maternal health. In addition, we will strongly depend on the Service project for obtaining the samples from healthy pregnant women matched to our MS cohort for age of the mother, parity, and sex of the child.

HEALTH DISADVANTAGES

MOTHER IS MORE VULNERABLE TO INFECTIONS

HIGH RISK FOR SEVERE INFLUENZA INFECTIONS

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Pregnant women are the highest risk group to develop severe influenza illness and even can die from it. This disadvantage for maternal health has been observed during so-called pandemics, the last one known as Mexican or Swine flu in 2009. Here, women had a 5 times higher risk to be admitted to hospital, compared to non-pregnant women. Therefore, vaccine recommendations have been revised worldwide and the World Health Organization now recommends vaccination of pregnant women as a first priority.

The mechanisms underlying this increased influenza risk for pregnant women are still largely unknown. Therefore, we aim to understand whether the influenza virus may be altered when a pregnant individual is infected. We also want to understand how the maternal immune system during pregnancy responds to the influenza virus and compare this to the immune response of non-pregnant individuals.

The risk to suffer from influenza during pregnancy can be reduced, if not prevented, by a vaccination against influenza. Surprisingly, only a small percentage of women during their reproductive years take advantage of this prevention method. Therefore, we anticipate that a better understanding of how pregnant individuals are at higher risk to suffer from severe influenza will increase the threat awareness of women during their reproductive years and therefore will contribute to a higher vaccination rate. Moreover, we expect that our identification of why the immune response to eliminate the influenza virus during pregnancy is weak will also be of relevance for other groups of people with a high influenza risk, such as the elderly, children, or patients taking immunosuppressive drugs.

PRENATAL STRESS CHALLENGE DISADVANTAGES FOR CHILDREN

IMPAIRED FETAL IMMUNE DEVELOPMENT

HIGHER RISK FOR IMMUNE DISEASES IN THE CHILDREN

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Prenatal stress challenge has been linked to disadvantages for children's health later in life, such as an increased risk for chronic immune diseases. The immune system largely develops during fetal life and hence, fetal immune organs may be a vulnerable for prenatal stress challenges. In turn, the impaired fetal immune development may become clinically evident during childhood by the onset of a chronic immune disease. Despite this growing recognition of an increased risk for chronic immune diseases in children in response to prenatal stress, the identification of pathways causally involved in this association is largely missing. This was the motivation for us to develop the present project. We here focus on the role of key stress hormones and glucocorticoids on fetal immune and organ development.

The fetus requires glucocorticoids in order to ensure structural and functional development of organs, such as the lung or immune organs. Surprisingly, until late in gestation, the fetus itself is not capable of producing glucocorticoids on its own. Therefore, maternal glucocorticoids are transported via the placenta to allow fetal development even in the absence of fetal glucocorticoid production.

However, the fetus must also be protected from overshooting maternal glucocorticoids, which are secreted in response to stress challenge. A placental enzyme called 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) acts as a ‘gatekeeper’ in order to protect the fetus.

Given the importance of this ‘gatekeeper’, what happens if 11βHSD-2 function is failing? This is what we aim to address in the present project. We seek to analyze mice that are genetically engineered to lack 11βHSD-2 exclusively in the placenta. This means that the fetus is unprotected from maternal glucocorticoids. This approach allows us to test the impact of overshooting glucocorticoids on fetal development compared to control mice. Moreover, we seek to understand the role of pregnancy hormones, for example progesterone, on strengthening the gatekeeper function of 11βHSD-2. We will compare our findings of glucocorticoids produced by the mother, the so-called endogenous glucocorticoids, on fetal development to the impact of exogenously given glucocorticoids and other medications taken during pregnancy, which are studied in projects titled “pain relievers during pregnancy” and “prenatal steroids”.

Our basic science approach involving mice allows us to dissect the importance of normal and extremely high levels of glucocorticoids on fetal development. In close collaboration with the Prenatal Determinants of Children’s Health study, in short PRINCE study ( www.uke.de/prince ), we will then be able to understand the relevance of our insights from mouse models for humans. We will test how mothers with high levels of stress perception and associated high levels of glucocorticoids can be identified. Once this is possible, we seek to develop strategies to prevent human fetuses from high glucocorticoid expose and risk for immune diseases later in life.

PRENATAL MEDICATION WITH ACETAMINOPHEN

IMPAIRED FETAL IMMUNE DEVELOPMENT

HIGHER RISK FOR IMMUNE DISEASES IN CHILDREN

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While fever and general pain are debilitating especially during pregnancy, medication might come along with long-term side effects for the child. Although a multitude of pain relievers are available, only few are considered to be safe during pregnancy. The most recommended agent is N-acetyl-p-aminophenol, acetaminophen, or APAP in short. It has been firstly used in 1893 and subsequently became a first-line medication for treatment of pain and fever without any obvious complications.

APAP is an OTC drug, meaning that it is available without prescription by a physician. Presently it is sold under different brand names, pure or in combination with other active compounds like vitamin C or caffeine. Since the 1990ies, APAP fell into disrepute because product label confusion resulted in unintentional overdosing, leading to cases of severe acute liver failure, especially in the US.
However, being apparently safe at therapeutic doses and being neither an opioid nor an inhibitor of coagulation, APAP remains the first choice to manage fever and pain during pregnancy.

So, why do we care about this obviously helpful drug?

Recent scientific studies showed, that APAP does more than relieving pain and reducing fever. The compound is able to alter our immune response. Being pregnant and taking this drug bears the risk of causing alterations in the immune system of the unborn child, too. This may lead to long term consequences like increased risk for asthma and probably other auto-immune diseases in the child. Some of the symptoms might show up not before their 7^th birthday.

With this knowledge we want to learn more about the mechanisms APAP works and especially how exactly our immune system is affected. In recent studies we showed that hormone levels were altered and that stem cell numbers in the developing child were reduced.

It is yet unknown how exactly APAP interacts with the developing fetal organs and why those long-term consequences might develop. Our project with focus on pain relievers during pregnancy therefore aims at developing a better understanding of how cells in pregnant women and their children react when they are treated with APAP.

We are following a modern bench-to-bedside strategy, designing models and experiments to mimic the complicated processes during pregnancy as close as possible. We are carefully analyzing the impact of APAP and its well-known toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) specifically looking at changes occurring in blood cells and in tissue. Using high tech multicolor flow cytometry we aim to identify different stem cells and immune cells, which are likely to be affected by the drug.

Our approach is embedded in harnessing the scientific ability of a consortium of partners within and outside the . Discussing ideas, conducting experiments and exchanging insights, in particular with the other projects dealing with prenatal challenges like endogenous and exogenous glucocorticoids, as well as the enables us to shed light on stem cell biology and immune during pregnancy. This will help us to understand how APAP affects the pregnant mother, the developing baby, and in the children later in life.
Combining our data we want to derive a recommendation for APAP medication for pregnant women and their physicians.

If you have any questions regarding this project feel free to contact us.

PRENATAL TREATMENT WITH STEROIDS

IMPAIRED FETAL IMMUNE DEVELOPMENT

HIGHER RISK FOR IMMUNE DISEASES IN CHILDREN

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Over the past 30 years, prenatal corticosteroid treatment, called betamethasone has been administered to thousands of women at risk of premature birth to accelerate fetal lung maturation in the preterm neonates. A remarkably higher survival-rate of premature and infants with very low birth-weight, highlights the efficiency of this treatment. However, the long term effects have not been analyzed in detail, especially the possible effects on the immune system. This is very important to know considering that other steroids are known to induce cell death of developing T cells.

The development of the immune system of mammals begins before birth. The thymus is an essential organ for T cell development and establishment of tolerance. At birth, thymic function is at its peak and therefore events affecting the thymus may have consequences for the child’s immunity as immature thymocytes are extremely sensitive to steroids. Injection of physiological doses of betamethasone has been shown to result in a dramatic reduction of the thymus volume and death of T cell precursors in our model . The lost niche is quickly replenished, and this accelerated maturation may result in errors during selection events. Of notable significance, in vitro treatment of human thymocytes with low betamethasone doses has also been shown to induce cell death of the developing cells.

Western Societies have been faced with an unparalleled increase in the incidence of autoimmune diseases and asthma over the past decades. In this context it is particularly noteworthy that the age at which diseases like type I diabetes occur is decreasing, and in many cases occur with more severe course, when compared to a few decades ago. Recent evidence indicates that the exposure to prenatal steroids is a risk factor for neonatal sepsis and for developing asthma and diabetes later in life. Given the importance of T cells in adaptive immune responses, we hypothesise that prenatal steroid treatment impairs the normal development of the offspring’s immune system and therefore increases the risk for autoreactivity/allergy later in life.

The main focus of our project is to study T cell development and long-term effects on the immune system in offspring whose mothers were treated with prenatal corticosteroids. To test our hypothesis we plan to apply our model of autoimmunity and allergy, and to analyse in detail the immune system at birth and later in life of children whose mothers received steroids.

In consideration of that half of the women receiving prenatal steroids have not delivered by the time that the beneficial effect of steroids wears out, awareness of harmful long term effects could lead to a re-evaluation of treatment protocols.

Western Diet: Hypothetical impact
on immune system and foetal and offspring's development

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Unprecedented consumption of high sugar, fat and salt dietary components, commonly referred to as „Western“ diet, is currently observed not only in developed countries, but also in developing countries and is considered one of the leading causes of obesity. It is apparent that a growing number of individuals are “forced” to consume this diet due to socioeconomic circumstances. In the year 2014, according to the World health organization, 600 million adults suffered from obesity. Many of them were also diagnosed with related metabolic or cardiovascular diseases. Interestingly, obesity is often associated with chronic and pathological activity of the immune system. Furthermore, epidemiological data indicate that consumption of Western diet during pregnancy and maternal obesity is often associated with chronic and pathological activity of the immune development and account for poor children’s health later in life.

This project aims to elucidate the cellular and molecular mechanisms underlying these effects. The main hypothesis is that Western diet and maternal obesity skew the maternal immune adaption to pregnancy, especially the CD4 T cell response required to mount tolerance towards the semi-allogeneic foetus towards inflammation. In turn, this inflammation leads to impaired foetal development and has a pivotal impact on the compositon of the intestinal mircrobiota, which in turn is in continuous cross tall with the immune system, we will test if diet-mediated inflammation is mediated by the Western diet mediated imbalance of the maternal microbiota.

Novel multi-cytokine fate reporter and conditional knock out mouse models will be used to test if maternal Western diet affects foetal development and offspring’s immunity via an altered function of distinct CD4 T cell subsets. To understand the molecular details of diet-induced changes in these mice, we will employ single cell trancriptomics, epigenetics, and state-of-the-art computational approaches. To translate the mechanistic insights from the mouse models into similarities and differences in humans, we will use data and biological samples from the prospetive pregnancy cohort PRINCE. Our project will help to identify new therapeutic targets such as pathogenic CD4 T cells, to develop new therapies, which aim to prevent the offspring having to suffer life-long health problems due to the social circumstances in which they are born.

Congenital Cytomegalovirus infection

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Congenital Cytomegalovirus infection

Cytomegalovirus (CMV) is a large double-stranded DNA virus and member of the Herpesvirus family. It is highly prevalent worldwide, with seroprevalence rates ranging from 40 to 100%. Primary infection is usually subclinical in healthy adults due to the control by the immune system. T lymphocytes are known to play a critical role in the control of CMV infection. However, the antiviral immune response cannot eliminate the virus, nor can it reliably prevent superinfection with additional CMV strains or reactivation of the latent infection.

Importantly, congenital infection with CMV in newborns is the most frequent cause of permanent disabilities including neurological damage, such as sensorineural hearing loss and mental retardation. Approximately 1% of children worldwide are born with congenital CMV infection, and ~10% of them are clinically symptomatic at birth and likely to suffer from long-term sequelae. Another 5-10% develop late-onset disabilities.

The factors determining vertical transmission from mother to foetus and the symptomatic versus asymptomatic outcome of congenital or neonatal CMV infection remain unknown. Additionally, there is no vaccine available and the only accepted treatment with Valganciclovir carries the risk of serious adverse effects.

In this project we aim to investigate factors that could increase the risk of prenatal CMV infection and severity of neonatal disease. We will determine the role of prenatal challenges, the anti-CMV T cell response of mother and child, and viral pathogenicity factors. We will investigate if maternal stress and medication intake during pregnancy – challenges which are potentially avoidable – render the offspring more prone to develop a severe course of early life CMV infection. In depth characterisation of maternal and neonatal antigen-specific T cells will define their role in control of CMV infection during the early life period. Finally, we will investigate the pathogen itself to identify viral genetic factors that determine its pathogenicity and likelihood of vertical transmission. In summary, we intend to answer pending questions of CMV infection biology that could lay the basis for new diagnostic or therapeutic approaches.

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Prof. Dr. Wolfram Brune

Head of Research Department Virus-Host Interaction

Heinrich Pette Institute
Leibniz Institute for Experimental Virology
Martinistr. 52
20251 Hamburg
Germany

Email wolfram.brune@leibniz-hpi.de