Principle Investigator

Scientific interest within the context of the graduate college:

Current preventive efforts mainly focus on the treatment of traditional cardiovascular risk factors (e.g. hypertension and diabetes). However, it is widely known that poor diet is a main risk factor for cardiovascular disease (CVD) that impacts both, gut microbial community structure and metabolic output. Yet, the specific food components promoting CVD and the underlying mechanisms remain for the most part elusive. This lack hampers effective public policy-making to refine primary prevention and improve resilience against CVD and associated complications.

Figure 1. Kaplan Meier plots for MACE stratified by xylitol quartiles (left). Forest plot for HR for MACE +/- adjustment for traditional CVD risk factors (right).

Artificial sweeteners have been widely introduced into the food chain over the past decades due to lower calorie content than sugar and presumed health benefits.

In particular, their use is recommended by multiple health bodies for patients with cardiometabolic disease. Though they are generally recognized as safe (GRAS) by regulatory agencies (e.g. US Food and Drug Administration and European Union), little is known about the long-term health effects of artificial sweeteners. We have recently found that artificial (including non-nutritive and low-calorie) sweeteners (e.g. erythritol and xylitol) are clinically linked with thrombotic event risk in large observational cohort studies and foster prothrombotic phenotypes in vitro (Figure 1, 2) and animal models of arterial injury.^1,2 Notably, some of the sweeteners are also produced as low-abundance intermediates in the human glucose metabolism and are therefore present in every patient irrespective of artificial sweetener use.

Figure 2. TRAP6-induced platelet-Leukocyte aggregates in whole blood are increased by xylitol.

Project description:

Introduction: Based on our preliminary and recently reported large-scale clinical observational studies and mechanistic cell-based and animal model studies, we hypothesize that endogenous production and consumption of commonly used sugar substitutes induce a pro-thrombotic and pro-inflammatory state.

In this project, we will first characterize pro-thrombotic and pro-inflammatory effects of commonly used sweeteners (endogenous and dietary non-nutritive and low-calorie sweeteners) in vitro. This approach will be guided by preliminary data from our epidemiological studies where we identified CVD risk thresholds for endogenously produced sweeteners as well as pharmacokinetics studies exploring post-prandial levels of dietary sweeteners following a typical dietary exposure.^1,2

In parallel studies, we will quantify direct effects of sweeteners on the platelet phenotype in humans using a prospective interventional study design. In collaboration with the clinical research unit of the Department for Endocrinology of the Charité Campus Benjamin Franklin patients at CVD risk will be enrolled.  Acute and chronic effects of sweetener consumption on vascular inflammation and thrombosis potential will be assessed. Participants will receive either a placebo control or candidate sugar substitutes. The study design was informed by our recently conducted clinical trials that tested pro-thrombotic effects of the polyol sweeteners erythritol and xylitol (Figure 3).^2,3

Figure 3. ADP-induced aggregation responses in healthy subjects before and after xylitol ingestion.

Aim 1: To test the hypothesis that commonly used sugar substitutes (sucralose, aspartame, saccharine, acesulfam-K, stevia, erythritol, xylitol, sorbitol) impact thrombosis-relevant phenotypes in vitro. We will perform gold standard methods for platelet functional testing, including light transmission aggregometry, flow cytometry, shear flow experiments, immunofluorescence microscopy and imaging flow cytometry.

Aim 2: By using a prospective intervention study design, we will test the hypothesis that acute and chronic exposure to sweeteners alters thrombosis potential in patients at CVD risk. Ex vivo platelet phenotypic changes will be monitored using the above-mentioned methods combined with metabolomics and proteomics studies to explore sweetener-induced metabolic alterations.

Overall, the proposed studies in Aim 1 and 2 will elucidate pro-thrombotic and pro-inflammatory effects of commonly used artificial sweeteners on the vascular phenotype. The results will provide important information to inform public policy-making for CVD prevention and improve the understanding of how sweetener consumption causally contributes to major adverse cardiovascular event risk.

References

Principle Investigator

Scientific interest within the context of the graduate college:

The most exciting phrase to hear in science, the one that heralds new discoveries, is not ‘Eureka!’ but ‘That’s funny…’   (Isaac Asimov)

We are a reproductive immunology group studying pregnancy, allergy, the gut microbiome and immune system development. Our research focuses on the “developmental origins of health and disease” hypothesis, which posits that perinatal environmental exposures (during the fetal and early neonatal life stages), can influence immunity and subsequent disease susceptibility later in life. Inappropriate nutritional inputs in early life can result in metabolic malprogramming of vital regulatory pathways which permanently alter the immune system, resulting hyperreactive inflammatory processes that persist for the lifetime of an individual.¹ As an example of this, childhood overweight and obesity present a serious public health concern, and are associated with an increased risk for non-communicable diseases such as asthma.² To address the increasing morbidity and mortality from non-communicable diseases, it is therefore crucial to understand the mechanisms that contribute to this phenomenon. The aim of this proposal is to use a mouse model to understand how overweight during infancy alters developmentally programmed inflammatory responses, as well as pathophysiological effects in the lung, with a focus on allergic asthma. The knowledge generated from this work will contribute to our understanding of how early onset metabolic derangements such as overweight, contribute to pathogenic changes in postnatally developing organ systems. This approach will identify potential pathways and windows of opportunity whereby we can counteract pathogenic developmental processes.

Project description:

Introduction: For your project, you will use our established mouse model of PostNatal OverFeeding (PNOF), in which mouse litters that are culled at 4 days of age encounter increased milk availability and excess calorie intake, and thus become overweight during infancy (an effect that persists into adulthood). We have previously shown with this model that PNOF mice have increased asthma severity, and your project will delve into the mechanism of why this is occurring. To do this your project will involve the following aims:

Aim 1: Characterize of how litter culling affects breast milk nutritional composition, as well as offspring body composition and serum parameters.

Aim 2: Assess the effect of early life overweight on the gut microbiome using metagenomic sequencing of fecal samples.

Aim 3: Understand how early life overweight affects immune system development and by performing 18-color flow cytometry in various organs.

To accomplish this, you will be trained in all aspects of mouse handling and reproductive strategies in our PNOF model, and also receive training in induction of experimental allergic asthma and analysis of the asthma phenotype. In addition to learning animal work and basic molecular biology techniques in the laboratory, your project will include major methods, such as gut microbiome metagenomic sequencing and 18-color flow cytometry immunophenotyping of different tissues.

References

Principle Investigator

Scientific interest within the context of the graduate college:

We study development and function of the innate immune system, in particular of innate lymphoid cells (ILC). A current focus is to obtain a molecular understanding of how the innate immune system, by integrating environmental signals (such as those derived from nutrients, microbiota, circadian rhythm) contributes to tissue physiology. Recent studies have revealed ever more intriguing relationships between innate immune system components and basic developmental and biologic processes that are likely to reveal unsuspected pathways by which the immune system might be plumbed to improve health and healthspan. These lines of research have suggested new functions of the immune system for processes such as tissue homeostasis, morphogenesis, metabolism, regeneration and growth. Our research is developing by crossing boundaries of disciplines (immunology, microbiology, developmental biology, stem cell biology, nutrition sciences, tumor biology, regenerative medicine etc.) and is, by nature, highly interdisciplinary.

Project description:

Introduction: Innate lymphoid cells (ILC) are tissue-resident innate lymphocytes that are involved in immunity to infections (Witkowski et al., Nature 2021; Hernandez et al., Nat Immunol. 2015) but are also deeply integrated in the regulation of tissue function. Based on our preliminary and on published data (Gronke et al., Nature 2019; Guendel et al., Immunity 2020; Diefenbach et al., Immunity 2020), we hypothesize that ILC regulate the function of non-hematopoietic cells to adapt organ function. In this project, we are exploring the role of ILC3 and of IL-22 in liver regeneration. Our preliminary data show that liver regeneration is dependent on IL-22. We have already obtained a high resolution scRNAseq atlas of the regenerating liver of wildtype mice and of IL-22-deficient mice that reveal molecular network of IL-22-dependent regeneration.

Three key questions will be addressed:
(1) How is IL-22 production regulated during liver regeneration? Preliminary data reveal a neuro-ILC-hepatocyte axis that controls hepatocyte differentiation and renewal.
(2) Which key regenerative pathways in hepatocytes are controlled by IL-22? Key data indicate that IL-22 signaling promotes a Wnt-driven regenerative program.
(3) Can IL-22 be used to promote liver regeneration? We use mouse genetics combined with CRISPR/Cas9-driven lineage tracing/barcoding and high dimensional single cell genomics.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our laboratory focuses on the immunological and molecular pathomechanisms of the skin with the aim to identify approaches for personalized and also preventive medicine. One of our translational research interests are chronic inflammatory autoimmune diseases of the skin. In addition to preclinical models and in vitro methods, we use skin and blood samples from patients for biomarker identification. We are particularly interested in specialized T cell responses, cytokine signaling pathways and the application of liquid biopsy in chronic inflammatory skin diseases.

Project description:

Introduction: Cell-free DNA (cfDNA) refers to DNA fragments that are found in body fluids, such as blood. In psoriasis, the release of DNA from damaged skin cells into the bloodstream is an active field of research for the implementation as a potential biomarker.^1-3 Thus, cfDNA may carry information about the inflammatory processes and cellular damage occurring in the skin.

The field of biomarkers in psoriasis is broad and continually evolving, with scientists aiming to identify reliable markers that can help in diagnosis, prognosis, treatment response assessment, and understanding the underlying mechanisms of the disease. We are interested in the physical and molecular features of cfDNA fragments to study treatment response and reconstitution of health.

Aims: The research project aims to validate the possibilities and limitations of cfDNA in psoriasis. This includes the quantification of cfDNA in the plasma of psoriasis patients as a marker for increased cellular turnover. The determination of the absolute copy number of psoriasis-associated genes of inflammatory activity (TNFa, IL17A/F, IL23, IL6) and epidermal activity (KRT16, KRT6, KRT17) as markers for disease activity. Within the framework of the doctoral project, patients with an active disease before the initiation of therapy are examined as well as those undergoing therapy (therapy responders and non-responders). The investigations are carried out in comparison with control subjects without inflammatory or autoimmune skin disease, or any other chronic disease such as cancer.

In addition, we aim to improve an assay that can accurately determine the amount of cfDNA originating from epidermal keratinocytes. For this purpose, the methylation of cfDNA from psoriasis patients and age- and sex-matched controls will be analyzed. With the help of the methylation atlas by Zhu et al. (Nat Methods, 2022)⁴ and Loyfer et al. (Nature, 2023)⁵ and is possible to perform a deconvolution of cfDNA that allows the composition with regard to the tissue origin.

The doctoral thesis is embedded in a pilot study that examines 20 controls and 120 patients with psoriasis as proof of concept. The focus of the doctoral thesis is the definition of qualitative and quantitative differences of cfDNA in psoriasis patients and healthy individuals, as well as treatment responders and non-responders. The overall goal is to validate the possibilities and limitations of cfDNA as a biomarker for disease activity, treatment response and flare prognosis in psoriasis.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our group is interested in immune and kidney epithelial cell interactions. Using analysis of cells in the urine, kidney biopsies and cell culture we focus on analyzing human patient samples to answer the overarching question, how the kidneys are damaged and how they recover from injury.

Project description:

Introduction: Kidney damage, in the acute form as acute kidney injury (AKI) or the chronic form as chronic kidney disease (CKD), is among the most frequent forms of organ damage. AKI and CKD both have a tremendous impact and are associated with increased morbidity and mortality, having a large negative impact on global health. In the proposed project we are interested in cellular mechanisms protecting us from kidney injury. The hypothesis of the proposed project is based on our previous observation, that immune and kidney tubular epithelial cells can be found in the urine and correlate with damage in AKI and inflammation in CKD.^1,2 These cells phenotypically resemble cells in the kidney, and can be used as a “window into the kidney” to understand pathophysiological processes in the tissue.^1,3 Interestingly however, increased amounts of immune and tubular epithelial cells can also be observed in critically ill patients without AKI and also in stable CKD patients, without being associated with damage. At present it is unclear, whether these cells just reflect subclinical damage, or whether they reflect an active adaptation to kidney stress. Therefore, in the proposed project we will use a combination of state-of-the-art methods such as single-cell sequencing (scRNAseq) and flow cytometry, aiming to understand which active cellular adaptation protects us from kidney damage. All required cohorts and techniques are established in our group.

WP1: Determine the gene expression of kidney tubular epithelial cells in patients without kidney damage. In this WP we will use scRNAseq of immune and kidney tubular epithelial excreted in the urine of patients without kidney damage. The patients will include critically ill patients without AKI and patients with stable CKD. The respective data will be compared to our existing data sets from patients with active kidney damage (AKI and different forms of other kidney diseases). This WP will yield differences in cell composition and gene expression between active kidney damage and stable kidney function.

WP2: Derive a flow cytometry panel from scRNAseq data. Based on the differences in cell composition and gene expression in WP1, we will establish an antibody-based staining panel to detect the respective differences using flow cytometry. This will enable us to analyze a larger cohort of patients in the subsequent WP.

WP3: Active and failed adaptation to kidney stress. In the last WP we will use flow cytometry and the staining developed in WP2 to assess a larger cohort of patients with kidney stress over time. The cohort will include CKD patients with and without stable kidney function as well as critically ill patients with and without AKI.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Health and disease is not well defined for tuberculosis, one of the most important infectious diseases. Here, we propose to build upon an existing cohort study of tuberculosis contacts in Berlin to define a recently recognized new form of TB, subclinical TB, using a novel phage-based assay. People with evidence of subclinical TB will be high-priority contacts for prophylactic anti-TB treatment to prevent progression to active TB.

Project description:

Introduction: Tuberculosis (TB) and its etiologic agent, human-adapted Mycobacterium tuberculosis (Mtb) complex, is the leading cause of death from a single infectious agent and the 13th total leading cause of death worldwide. Overall, an estimated 1.8 billion people, 20-25% of the world’s population, is infected with Mtb, yet the majority of infected individuals do not fall ill to TB. Conventionally, TB is classified into active disease vs. latent TB infection. Active disease is defined by clinical symptoms (fever, cough, weight loss), radiological signs (cavities), and microbiological proof of disease (PCR or growth in culture). Latent TB (LTBI) is defined as immunoreactivity to specific TB antigens, most commonly through Interferon Gamma Release Assays (Quantiferon Gold In Tube) or more traditionally, by tuberculin skin testing in the absence of the aforementioned signs. In recent years, there has been growing recognition that the binary classification of TB into “latent” and “active” does not accurately reflect the complex pathophysiology of Mtb infection and the processes that lead from health to disease. The binary classification may be inadequate for informing research and programmatic advances for global TB elimination.¹ In contrast, subclinical TB has been used to refer to a disease state where individuals are asymptomatic, but infectious (Figure 1).² Studies estimate that up to 68% of global TB transmissions are due to subclinical TB.³ Subclinical TB thus represents a key stage of TB that sits between health and disease and that is challenging to diagnose in the absence of clinical symptoms. Better methods are thus needed to detect individuals with subclinical TB.

The Actiphage method uses a bacteriophage that is specific for the Mycobacterium genus. The phage is used to efficiently lyse bacterial cells present in a sample to release mycobacterial DNA making it accessible for detection by PCR, using primers mycobacterial species or groups-specific primers (i.e. IS6110 insertion sequences). Importantly, the bacteriophage method only allows for the detection of viable mycobacteria, as viable bacteria are required for the phage to replicate and then to lyse the host cell at the end of its replication cycle. Therefore this combination of phage and PCR provides enhanced sensitivity and specificity and live/dead differentiation due to the efficacy of the bacteriophage host-lysis process.

To perform the assay, peripheral blood mononuclear cells (PBMCs) are isolated from 2 ml of heparinized blood. The PBMCs are lysed by osmotic shock to release any intracellular mycobacteria. The bacteriophages are added to the sample and incubated for 3.5 hours to allow the phage to complete their replication cycle and lyse the mycobacterial cells. Any remaining intact bacterial cells are separated from free genomic DNA released by the phage by filtration. The DNA is then further cleaned and concentrated before any mycobacterial genomic DNA present in the sample is detected by specific PCR.

Figure 1. Disease strata in tuberculosis.⁴

For TB, the method has not been formally validated against other direct detection methods, because there are none that are as sensitive as the Actiphage test (levels of Mycobacterium tuberculosis in blood are normally too low to detect).  In cattle, measured against the comparative skin test [SICCT], the sensitivity of the assay was shown to be 98% with 100% specificity (n = 41 SICCT-positive animals; n = 45, SICCT-negative animals).⁵ In humans, the Actiphage blood testing had a sensitivity of 73.3% (95% confidence interval [CI] 48.1-89.1%) and a specificity of 100% (95% CI 56.6-100%).⁶

The overall objective of this project is to conduct a prospective clinical cohort study of contacts of TB patients and classify these individuals into TB infection (i.e. a positive IGRA), subclinical TB (i.e. a positive Actiphage test) and no clinical symptoms, or clinical TB (symptomatic). The study builds upon existing collaborations (Prof Cath Rees, University of Nottingham, Actipahge assay) and the Berlin TB contact cohort study with the Gesundheitsamt Berlin (PI Matthias Gröschel, recruiting contacts since Q1/2024). In this study, all contacts who provide written informed consent are included and undergo blood testing as part of their routine visits to the Gesundheitsamt.

Aim 1: Establish the Actiphage test in our labs at the Department of Infectious Disease at Charité – Universitätsmedizin Berlin. This will include a 2-week visit of the doctoral candidate to the Cath Rees lab in Nottingham to get acquainted with the method.

Aim 2: Determine the proportion of subclinical TB among exposed contacts in Berlin using the Actiphage test. We hypothesize that the test will detect subclinical TB in ~20% of exposed contacts and will help to further identify contacts who require preventative treatment. We also hypothesize that these contacts are at a higher risk of progressing to active TB. TB contacts will provide an additional blood tube for PBMC isolation. Contacts for this analysis will be recruited during three months (n = ~500 contacts) and will be contacted every three months for 1 year (80-90% of exposed contacts will develop active TB within the first year to ask for symptoms suspicious of TB).⁷ The aim will be to describe what proportion of contacts is IGRA positive (test is routinely performed already), Actiphage positive, and presents with symptoms of active TB.

The study will be further embedded into a larger study of sympatry, i.e. the preferential interaction of Mtb strains and their hosts. The role of subclinical infection is unknown at this stage. The prospective students will obtain critical skills in infection epidemiology by active engagement in the Berlin TB household study. The students will further learn basic lab skills, including preparation of clinical samples, isolation of PBMC and performing of Actiphage assays, along with PCR. Finally, prospective students will become acquainted with data analysis tools and statistics.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our group is interested in the identification and investigation of genetic, clinical, and environmental factors determining the onset of chronic kidney disease (CKD) and kidney survival. We make use of next-generation sequencing techniques and deep-phenotyping to identify genetic variants that are predictive for disease progression or convey protection from organ failure. We functionally evaluate identified germline variants in vitro in order to understand underlying molecular mechanisms leading to CKD on the one hand or protecting from kidney failure on the other. By doing so, we aim at defining and targeting molecular switches responsible for health maintenance and disease alleviation.

Project description:

Introduction: Nephrolithiasis, or kidney stone disease (KSD), is a common global healthcare problem with a lifetime prevalence of 10-20%. Kidney stones recur frequently and cause substantial morbidity –including CKD, reduced quality of life and enormous cost. Kidney stone formation is strongly influenced by genetic factors (positive family history in 30-60%) and >30 Mendelian forms of KSD were described. Yet, molecular mechanisms of stone formation remain unknown in many adult kidney stone formers (KSF). Increasing data suggests relevant genetic risk for KSD from heterozygous variants in genes traditionally believed to have recessive inheritance like SLC34A3,^1,2 a phenomenon that is called gene dosage. We recently performed exome sequencing (ES) of a large cohort of 787 adult KSF and 114 non-KSF to assess prevalence of genetic disease in adult stone formers. Thereby, we detected a relatively low prevalence of Mendelian disease, but enrichment of genetic variants predisposing to KSD, amongst others, a heterozygous variant in CLDN16,³ encoding a renal tight junction protein. Homozygous pathogenic variants in CLDN16 (Claudin 16) cause a severe disease manifesting in childhood – often leading to kidney failure.⁴ Data suggest that heterozygous variants in CLDN16 may cause a less severe, but intermediate phenotype associated with an increased risk of KSD and CKD.⁵

Aim 1/WP1: Identification of genetic variants with potential gene dosage effect relevant for KSD. The applicant will perform detailed genotype-phenotype correlation of variants in CLDN16 inthe given dataset and compare results with a validation cohort of 800 KSD and with data in public repositories (e.g., Genomics England, UK Biobank, AllofUs). A similar approach assessing other rare and common variants (identified by single variant analysis and aggregated variant burden analysis) will be conducted for other candidate KSD genes with suspected gene dosage effect to identify genes and variants suitable for functional analysis (WP2).

Aim 2 /WP2: Functional validation of genetic variants with potential gene dosage effect in KSD. Initially, heterozygous CLDN16 variants found with increased prevalence (e.g., enriched) in stone formers will be studied. In a second step, a limited number of candidate genes and variants identified in WP1 will be assessed. For CLDN16, variants will be characterized in vitro in an established overexpression system in collaboration with the laboratories of Prof. Dorothee Günzel and PD Dr. Jörg Piontek, both known experts in tight junction proteins. Additionally, depending on the results from overexpression studies, characterization of urinary renal epithelial cells isolated from selected individuals with the respective CDLN16 variants will be performed. Planned analyses include qRT-PCR, Western Blot, immunofluorescence imaging and measurement of transepithelial resistance (TER).

References

Principle Investigator

Scientific interest within the context of the graduate college:

Neuro-immune interactions.

Project description:

Introduction: Toxoplasma gondii, a ubiquitous intracellular parasite with a profound ability to infect virtually all warm-blooded animals, is responsible for a significant public health concern. In humans, infection can lead to toxoplasmosis, which, while often asymptomatic in healthy individuals, poses severe risks to immunocompromised patients and pregnant women.¹ Moreover, emerging evidence suggests T. gondii’s potential triggering of various neurological disorders, including schizophrenia and other psychiatric conditions, underscoring the critical need to understand the parasite’s interaction with the nervous system.² The central and enteric nervous systems are intricate networks that regulate numerous vital functions, from cognition and sensory processing in the central nervous system (CNS) to gastrointestinal motility and secretion in the enteric nervous system (ENS).³ T. gondii’s ability to invade and persist in these neural environments suggests significant adaptations by both the parasite and host neurons. Investigating these adaptations, particularly at the transcriptional level, provides insights into the mechanisms of pathogenesis, neuronal resilience, and potential neuropathology associated with infection.

We aim to monitor the transcriptional changes occurring in neurons during T. gondii infection. To this end, we have already established the respective lines and protocols for bulk sequencing of purified RNA from sort-purified GFP⁺ neuronal nuclei from the T. gondii-infected brain and gut tissue of the Snap25^Cre/+ x INTACT system, in which the GFP expression is only activated in neurons. To monitor alterations in enteric neurons’ composition and transcriptional changes in subsets of enteric neurons, we will perform RNA sequencing from brains and gut tissue. To this end, we will separately sort-purify GFP⁺ nuclei from Snap25^Cre/+ x INTACT system to obtain highly pure neuronal nuclei. These neuronal cell nuclei will be analyzed using the RNA-sequencing platform, which we have already implemented for single-nuclei sequencing. Together, these experiments will identify genes in neurons regulated in the context of T. gondii infection.

Based on our data detecting a type II interferon signature in the ENS in various inflammation models, we will perform bulk RNA sequencing of the ENS and CNS, in which we have deleted the Ifngr1 specifically in neurons by the Snap25^Cre/+ x Ifngr1^fl/fl INTACT system and which allow for sort-purification based on the nuclear GFP signal. This experimental setup will allow us to study the potentially protective gene expression signature in neurons induced by IFN-γ. Furthermore, these experiments will enable us measure if neuron-specific deletion of the IFN-γ receptor results in increased T.gondii burden in various organs. Therefore, these experiments will reveal how IFN-γ-signaling in neurons contributes to anti-toxoplasma immunity.

Taken together, these datasets will yield correlations of differentially expressed genes of ENS and CNS neurons and their relative abundances, exposing disease-relevant signaling circuits triggered in neurons. This global perspective is essential for uncovering the molecular underpinnings of T. gondii’s impact on neuronal function and survival, which could lead to the development of targeted therapies and interventions.^1,2

References

Principle Investigator

Scientific interest within the context of the graduate college:

The kidney has a central role in absorbing nutrients for tissue function and health. Vice versa, cellular toxins have to be excreted by the kidney into the urine to avoid systemic accumulation and toxicity.^1,2 Our working group recently described a patient who has a genetic defect of the sulfate transporter SLC26A1.³ SLC26A1 has been postulated to mediate sulfate absorption in exchange for oxalate. Sulfate is an important nutrient for musculoskeletal as well as cardiovascular health. Our patient described with a mutation of SLC26A1 has been suffering from a musculoskeletal disorder. Additional studies performed in large population cohorts (UK Biobank) have now shown that defective sulfate transport may be more common than expected and is related to bone fractures (manuscript in revision). We hypothesize that the anion exchanger SLC26A1 mediates sulfate (nutrient) absorption in the kidney for exchange of oxalate (toxin). In order to investigate this as part of a doctoral thesis, we have generated human pluripotent stem cells deficient for SLC26A1. The doctoral student will be culturing these stem cells to so called kidney organoids following a protocol established in our laboratory (Constantin Dickel, Re-Thinking Health, Class of 2022). The doctoral student will be performing transport studies using radioactive tracers and examine the effect of sulfate and oxalate transport defects on cellular toxicity (metabolomics, mitochondrial function).

Project description:

Introduction: The doctoral thesis will examine the hypothesis that SLC26A1 mediates sulfate absorption in exchange for oxalate using a kidney organoid model. In a first step, the doctoral student will be examining whether sulfate is taken up from kidney cells. In a second step, the doctoral student will be comparing kidney tubuloids from wild-type cells in comparison to cells deficient for SLC26A1. The doctoral student will be examining the effect of oxalate accumulation on cellular toxicity as measured by mitochondrial dysfunction. In case SLC26A1 mediates oxalate uptake from the basolateral membrane in exchange for sulfate, we predict that SLC26A1 deficiency will protect from cell death induced by oxalate due to impaired oxalate uptake into the cell. This may represent a novel pharmacological approach to protect the kidney from oxalate toxicity specifically for patients suffering from oxalate-related disorders. To this end, the following specific aims will be pursued:

Aim 1: Characterize sulfate and oxalate transport in kidney tubuloids from pluripotent stem cells.

Aim 2: Examine the effect of SLC26A1 deletion on cellular uptake of sulfate and oxalate.

Aim 3: Examine the effect of SLC26A1 deletion to prevent from oxalate toxicity and cell death.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our group “Signal Transduction in Health and Disease” (Department of Hepatology and Gastroenterology, Charité Campus Mitte and Campus Virchow) aims to understand the molecular mechanisms involved in tissue homeostasis, inflammation, and resolution of inflammation. Our main focus is on the transcription factor NF-κB and its role in the intestinal epithelium. Our current projects range from determining the role of the transcription factor in epithelial regeneration in colitis and in inflammatory bowel diseases (Re-Thinking Health, 2022), to refining analgesia (3R funded project), to investigating the role of NF-κB in cellular senescence in the gut (DFG) or its role in metabolism.

Project description:

Introduction: Senescent cells are characterized by terminal cell cycle arrest, epigenetic changes, and by the senescence associated secretory phenotype (SASP). SASP is comprised of scores of cytokines, chemokines, and growth factors, which regulate cells and tissues in a paracrine manner. Therefore, even a small number of senescent cells can have a dramatic impact on distant tissues and were shown to sustain inflammation, fuel carcinogenesis, and shorten lifespan. Most of the genes that code for SASP factors are transcriptionally regulated by NF-κB8.^1,2 Under physiological conditions SASP promotes wound healing and recruitment of immune cells, which help clear senescent cells.

Figure 1. Multiplex immunofluorescence staining of senescence. Mouse colon (colitis model) was stained with markers for senescence. Simultaneous staining with numerous markers (multiplex) is required to rule out other cell states (proliferation, apoptosis, quiescence). Zoom-in on select fully senescent cells is shown on the right.

We are interested in investigating what kind of senescent populations arise in inflammatory bowel diseases (IBD) versus those that progressed to colorectal cancer (CRC). We are especially interested in the role that SASP plays in disease progression and whether distinct forms of SASP can be exploited therapeutically.

As part of our preliminary data, we have already identified distinct populations dependent on stage of inflammation and importantly, we show that no single type of senescence exists, but rather cells display different proficiencies for SASP and thus for communication with their environment. It is not known how these differences skew progression of IBD.

Aim 1: Characterize senescent cells in IBD and CRC and in murine colitis and CRC models. As part of this work package you will perform multiplex stainings (as in Figure 1) using already available samples from IBD and CRC patient samples and from murine mouse models. You will also receive training in basic analysis of single-cell RNA sequencing and will therefore quantify cells that have SASP versus those that do not. You will then isolate these cells using FACS and determine how different types of SASP alter immune cell recruitment.

Aim 2: Reprogram senescent cells to transform immunosuppressive “cold” tumor microenvironment environment into “hot”. Here you will use available senolytics and senomorphics (drugs that kill senescent cells or suppress their SASP), and those identified via our collaborators through deep learning, to reprogram senescent cells in 2D culture and in human and mouse intestinal organoids. Successful senolytics will be used in pre-clinical trials in CRC mouse models to determine if these expose tumors to immunosurveillance.

Figure 2. Immunofluorescence staining of mouse fluorescent organoid. All our projects combine work with mouse models. patient samples (including organoids) and bioinformatics.

Methods you will learn as part of this project: multiplex immunofluorescent staining, single-cell data analysis, immunohistochemistry, human and mouse organoid cultures (Figure 2), qPCR.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Role of tissue-resident macrophages during the maintenance of tissue homeostasis and the prevention of inflammatory disorders.

Project description:

Introduction: Tissue-resident macrophages including brain microglia, synovial macrophages and alveolar macrophages are considered essential for the maintenance of tissue homeostasis and the functionality of the respective organs.^1,2 A common feature of these cells is their capacity to constantly ingest and remove dying cells and large amounts of cellular debris that accumulate during physiological tissue turnover and tissue damage, respectively. Dysfunction of this macrophage compartment thus results in the accumulation of cell-derived danger signals and eventually triggers the development of spontaneous inflammatory and degenerative diseases.^3,4

Our previous work demonstrates that the constant requirement of an efficient phagocytic capacity imposes a substantial metabolic challenge for tissue macrophages, suggesting that metabolic adaption is a key feature of these cells.^5,6 To better understand the functional and metabolic properties of tissue-resident macrophages during steady state and inflammation, we plan to assess the specific metabolic features of individual macrophage subsets in vivo and study the impact of their capacity to metabolically adapt for tissue homeostasis and maintenance of health. We will use cutting-edge approaches including single-cell sequencing, spectral flow cytometry and fluorescence lifetime imaging (FLIM) to perform a detailed characterization of the metabolic states of macrophage subsets in vivo. Macrophage-specific conditional deletion of TFAM, an essential mitochondrial transcription factor, will allow studying the relevance of metabolic adaption of macrophages for tissue homeostasis.

Aim 1: Analysis of the metabolic properties of macrophage subsets during steady state and inflammation Using single-cell RNA sequencing as well as flow cytometry in conjunction with FLIM, we will characterize the metabolic state of tissue-resident macrophages and monocyte-derived macrophages during steady state and pulmonary inflammation.

Aim 2: Understanding the role of metabolic adaption of macrophages for the maintenance of tissue homeostasis. A conditional deletion of the transcription factor TFAM in macrophages will allow to determine the contribution of mitochondrial function and the metabolic plasticity of macrophages during the maintenance of health and prevention of inflammatory diseases.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Pulmonary arterial hypertension is a cardiopulmonary disease that is characterized by profound remodeling of the pulmonary vascular tree and has a poor prognosis if left untreated. Current therapeutic approaches rely on vasodilatory drugs and do not address the underlying cause of vascular remodeling. Investigating the pathophysiology of pulmonary arterial hypertension on a novel, in vitro microvasculature-on-chip model allows to track changes of the vascular system and the dynamics of individual cell types specifically in a temporal and spatial context that was not possible before. As such, microvasculature-on-chip models – in combination with advanced imaging modalities, state-of-the-art transcriptomic and proteomic analyses, and functional read-outs – open up unprecedented avenues for the discovery of new disease mechanisms and therapeutic targets.

Project description:

Introduction: Pulmonary arterial hypertension is caused by extensive pulmonary vascular remodeling that results in increased pulmonary vascular resistance and pulmonary arterial pressure, eventually causing right ventricular dysfunction and, ultimately, right ventricular failure and death. Pulmonary vascular remodeling in pulmonary arterial hypertension is characterized by proliferation and hypertrophy of endothelial and smooth muscle cells in pulmonary resistance vessels, and a parallel loss of pulmonary microvessels, especially in the alveolar capillary network, termed microvascular rarefaction or pruning.

Recently, this microvascular rarefaction has been linked to a loss of microvascular pericytes. Pericytes are perivascular cells that are encased within the microvascular basement membrane and assist in the maturation and stabilization of microvascular networks. To this end, pericytes communicate with endothelial cells by both direct physical contact, via gap junctions and by paracrine signaling. This interaction promotes the stabilization of endothelial cells and the maintenance of vascular barrier function. Accordingly, loss or detachment of pericytes may result in increased microvascular leak and the disintegration of microvascular networks. Recent histological analyses in lung tissue samples from PAH patients and animal models of pulmonary arterial hypertension reported an increased density of pericytes in pulmonary arterial resistance vessels, while the abundance of pericytes in the pulmonary microvasculature was markedly reduced. Based on this finding, we and others have hypothesized that in PAH, pericytes may migrate from the pulmonary capillary bed to the proximal arteries where they may integrate into the arterial media as contractile cells. Such a process would not only promote arterial remodeling and muscularization, but may also in parallel destabilize the pulmonary microvasculature causing microvasculature rarefaction. The actual effect of pericyte loss on pulmonary microvascular networks has, however, so far not been elucidated due to the lack of appropriate models and methods.

Histopathological analyses in human tissue samples or animal models are typically limited to a single time point and as such, cannot assess the dynamics and interactions of distinct cell types over time and space, or track the process of microvascular rarefaction over time. In vitro assays, on the other hand, often fail to reflect the complex multicellular and physicochemical context of the intact lung. In the present project, we will apply a unique microvasculature-on-chip platform that we have successfully developed in close collaboration with microphysiological model experts in Switzerland for the study of pericyte-endothelial cell interactions in pulmonary microvascular networks, and that we will employ here to study the effects of a targeted loss of pericytes on pulmonary microvascular network stability and the associated lung endothelial phenotype. Specifically, we aim to realize the following research aims:

Aim 1: To establish a system for targeted pericyte loss in pulmonary microvasculatures-on-a-chip. Pericyte loss will be induced by batrachotoxin, an activator of voltage-gated Na⁺ channels that are present in pericytes yet not in endothelial cells. As such, batrachotoxin should cause sustained membrane depolarization and ultimately, cell death only in pericytes. Induction of selective pericyte cell death will be assayed in cultured human lung pericytes and pulmonary microvascular endothelial cells, as well as in our pulmonary microvasculatures-on-a-chip platform.

Aim 2: To study the effects of targeted pericyte loss on microvascular structure in a pulmonary microvasculatures-on-a-chip platform. To this end, selective pericyte cell death will be induced by batrachotoxin in pulmonary microvasculatures-on-a-chip generated from cultured human lung pericytes and pulmonary microvascular endothelial cells, and effects on microvasculature network morphology and function will be studied by real-time imaging and digital image analysis.

Aim 3: To study the effects of targeted pericyte loss on the transcriptomic and functional phenotype of microvascular endothelial cells in a pulmonary microvasculatures-on-a-chip platform. Following targeted pericyte loss, endothelial cells will be retrieved from pulmonary microvasculatures-on-a-chip and undergo transcriptomic analysis by single-cell RNA sequencing and phenotypic characterization.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Alarmins can be used as markers for pathologies even prior to obvious onset of disease. Moreover, interference with alarmins and their signaling pathways provides a powerful tool for the manipulation of several types of diseases (Liew et al., Nat Rev Immunol. 2016; Peine et al., Trends Immunol. 2016).

Project description:

Introduction: Recently we reported on the discovery of a type-1 immunity-restricted promoter of the IL-33 receptor gene ST2. Transcription of ST2 starting from this promoter is essential for CD8+ cytotoxic and CD4+ T-helper-1 cell responses to virus infection (Brunner et al., Nat. Immunol. 2024). During transcription of ST2 also a short isoform lacking the transmembrane domain is generated by alternative polyadenylation. This soluble IL-33 receptor sST2 is assumed to act as a decoy receptor for IL-33. However, its roles in osteoarthritis and type-1 versus type-2 T-cell responses remain enigmatic.

Aim 1: In this project we plan to use our newly generated mouse strains lacking the soluble IL-33 receptor to investigate the relevance of soluble ST2 for the generation and progression of osteoarthritis. Osteoarthritis will be induced in the relevant mouse strains either by surgery (destabilization of the medial meniscus (DMM) model) or spontaneously by aging of the mice (Shen et al., Proc Natl Acad Sci USA. 2023).

Aim 2: In another line of experiments we want to study the role of soluble ST2 in type-1 versus type-2 T-cell responses. Here the relevant mouse strains will either be infected with viruses to generate type-1 T-cell responses or they will be challenged by an airway inflammation model that triggers type-2 immunity. We will assess the effects on T-cell differentiation and expansion, viral clearance, and immunopathology.

References

Principle Investigator

Scientific interest within the context of the graduate college:

The microbiota, i.e. the entirety of microbes in an organism, plays a crucial role in the maintenance of health and the development of diseases. A diverse microbiota not only produces essential metabolites and supports the uptake of nutrients, but also is important for the development of a functional immune system.^1,2 Mice that are born and kept under laboratory, specific pathogen-free (SPF) conditions have only a reduced microbiome and thus, possess an immature immune system, resembling more likely a newborn than an adult human.³ This may have contributed to failure in translating results from previous preclinical studies into the clinics. However, mice are an invaluable tool in preclinical research, as their genome is well studied and various genetic models exist that are necessary to understand molecular mechanisms of disease pathogenesis. To improve translatability, Wildling mice have been developed that harbor a natural microbiome, comparable to mice in the wild, and consequently, have an immune system being more similar to that of an adult human.⁴ As part of the “Wildling Mice in Health and Disease (HeaD)”-Consortium at Charité we will validate the Wildling model in our preclinical mouse model of muco-obstructive lung disease, called βENaC-transgenic (βENaC-Tg) mice.⁵ These mice overexpress the β-subunit of the epithelial Na⁺ channel (ENaC) specifically in the airways to produce airway surface dehydration, resulting in the development of airway mucus plugging, dysbiosis, chronic inflammation and structural lung damage, i.e. key features of patients with cystic fibrosis (CF) and other muco-obstructive lung diseases.^5-7 Although this mouse model mimics airway surface dehydration as a key disease mechanism in CF lung disease, important clinical aspects like spontaneous Pseudomonas aeruginosa infections are not recapitulated in SPF mice. Hence, we hypothesize that a natural airway microbiome will critically affect the infection status in βENaC-Tg mice including the development of chronic Pseudomonas infection that is a key determinant of lung disease severity and lung function decline. Such a novel model of chronic Pseudomonas infection would enable in vivo testing of novel therapeutic strategies to eradicate Pseudomonas from the airways and thus, target a high unmet need in patients with CF.

Project description:

Introduction: The overall aim of this research project is to evaluate whether βENaC-Tg mice with CF-like lung disease that harbour a “wild”, natural airway microbiome will develop chronic Pseudomonas infections, which is currently the missing human phenotype in SPF βENaC-Tg mice, and consequently, will be of major advance for preclinical testing of novel anti-infective strategies. For this purpose, we will generate Wildling βENaC-Tg mice and the development of the airway microbiota throughout distinct developmental stages (neonatal, juvenile, adult mice) will be carefully followed by culture-dependent and -independent (16S rRNA gene sequencing) techniques of bronchoalveolar lavage (BAL) and lung homogenates (Aim 1). Further, distinct lung immune cell populations will be characterized (Aim 2), concomitant with the assessment of inflammation markers and airway mucus obstruction to study thoroughly the impact of a natural microbiota on the progression of chronic lung disease (Aim 3). Finally, transcriptomic analyses will be conducted to obtain insights into molecular mechanisms underlying the pathogenesis of muco-obstructive lung disease in presence of a natural airway microbiota. Through a direct comparison with SPF animals, these analyses will comprehensively evaluate whether Wildling βENaC-Tg mice will mimic more closely disease mechanisms in patients with muco-obstructive lung diseases, including the development of chronic Pseudomonas infection, which is a key determinant of lung disease outcomes in patients with CF, and would thus authorize in vivo testing of novel anti-infective therapeutic strategies. In addition to basic molecular biology laboratory work, this experimental MD thesis also applies state-of-the-art multi-color flow cytometry and sequencing technologies.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Chronically stimulated surfaces of the body, in particular the gastrointestinal (GI) tract, are major sites where immune cells traffic and reside. Since the intestinal surface is constantly challenged by fluctuating environmental perturbations (microbiota, diet, pathogens), immune cells at this site display a remarkable capacity to adapt their functionality in order to safeguard organ homeostasis and health.

The Neumann lab is specifically interested in the molecular basis of immune cell adaption to the GI tract. The goal of our research is to understand the molecular mechanisms that determine the gut-specific functions of distinct immune cell populations. Furthermore, we aim to identify the specific (micro)environmental cues that trigger adaptation of immune cells in the gut. In addition, a major focus of our research lies on the crosstalk between gut immune cells and distinct intestinal tissue cells, such as epithelial or neuronal cell populations, to better understand the cellular networks that are in place to establish and maintain intestinal health.

Project description:

Introduction: The GI tract is equipped with the largest collection of neurons outside the brain, known as the enteric nervous system. Consequently, neuroactive substances, such as endogenous opioid peptides that are synthesized by the human body, can activate opioid receptors on the enteric circuitry to control crucial physiological functions such as gastric emptying and intestinal motility. Clinically, the importance of opioids in the gut is illustrated by the side effects associated with pharmacological opioid intervention during pain therapy, such as Opioid-induced bowel dysfunction (OIBD) or constipation. Vice versa, therapeutic administration of exogenous opioids is widely used to manage severe diarrhea as well as irritable bowel syndrome (IBS). Thus, the amount of bioavailable opioid peptides in the GI tract is strictly determining gut function and health. In addition, endogenous opioids have immunomodulatory functions and can counteract inflammation-associated pain, making them prime therapeutic targets during (intestinal) inflammatory diseases. Intestinal Foxp3+ regulatory T cells (Treg cells) are key for intestinal tolerance induction and host defense by actively controlling immune responses towards dietary and microbial antigens as well as invading pathogens. In addition to these cardinal immune-related roles, intestinal Treg cells also exert important non-immune functions in the gut, such as promoting local tissue repair and preserving the integrity of the epithelial barrier. Importantly, recent data obtained from mouse models in our lab have generated the hypothesis that intestinal Treg cells are also prominent producers of endogenous opioids in the gut. Thus, this project aims to better characterize and functionally understand these opioid-producing Treg cells.

Aim 1: Phenotypic, functional and spatial characterization of opioid-producing intestinal Treg cells. Intestinal Foxp3+ Treg cells are a heterogenous cell population, comprising of distinct subsets with different developmental origins, functions and phenotypes. Therefore, in Aim 1, we plan to thoroughly characterize murine opioid-producing gut-resident Treg cells in depth by multi-parameter flow cytometry and fluorescence-activated cell sorting (FACS). In addition, with the help of immunofluorescence microscopy, we aim to determine the precise location of opioid-producing Treg cells within the gut tissue, especially in close vicinity to e.g. enteric neurons. Thus, Aim 1 will be instrumental to obtain a detailed phenotypic, functional and spatial mapping of opioid-producing Treg cells and their potential interaction partners in the mouse gut.

Aim 2: Identification of signals regulating opioid-producing intestinal Treg cells. To identify the regulatory circuits that control the expression of opioids by intestinal Treg cells, we will systematically test potential signals (e.g. immune signals, dietary signals, microbial signals) during in vitro cultures or with the help of transgenic knock-out mice. Thus, Aim 2, will define the unique signals that induce and control the expression of endogenous opioids by gut-resident Treg cells.

Aim 3: Analysis of the functional role of opioid-producing intestinal Treg cells. To ultimately test the functional role of opioid-producing Treg cells, we have generated conditional Treg cell-specific opioid knock-out mice. In Aim 3, we will use these mice to perform functional analyses testing neuro-dependent gut functions, such as gut motility, peristalsis or secretion. Furthermore, we also aim to characterize the impact of opioid-deficiency on the phenotype and function of the enteric nervous system.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Disease prevention is taking a central role in tackling the burden of cardiovascular disease in our aging society. Thrombotic cardiovascular diseases such as venous thromboembolism, stroke and myocardial infarction are main drivers of morbidity and mortality worldwide. A central pathomechanism in these thrombotic cardiovascular diseases is termed thromboinflammation and arises from a deleterious dysregulation of an evolutionary conserved host defense mechanism involving platelets, the innate immune and coagulation system. There is an unmet clinical need for a better mechanistic understanding of thromboinflammation, in order to novel regulatory mechanisms that may pave the way for potential therapeutics, biomarkers and preventive measures. By pursuing this aim we established an across-species approach to investigate the paradox that long-term immobility during hibernation in brown bears and patients with spinal cord injury (SCI) does not increase the risk of thrombosis.

Our research acts at the interface between immunology, cardiovascular science and preventive medicine by taking advantage of an interdisciplinary translational research approach.

Project description:

Introduction: In this project we will characterize long-term immobility-induced thromboprotective mechanism arising within the innate immune cell compartment (i.e. neutrophils and monocytes) as key player in thromboinflammation and thrombotic cardiovascular diseases.

In the initial phase of venous thrombus development, primed monocytes expose TF to induce the extrinsic coagulation pathway while activated neutrophils release NETs as prothrombotic scaffolds triggering activation of the intrinsic pathway that both culminate in excessive fibrin formation. This is paralleled by release of neutrophil elastase that degrades TF pathway inhibitors thereby suspending antithrombotic mechanisms and aggravating thrombus formation. However, during later stages of thrombosis, neutrophils regulate thrombus resolution by modulating plasmin-mediated fibrin degradation and metalloproteinase-mediated reorganization of the thrombus matrix. In preliminary data we found that indicate that central neutrophil functions, such as bacterial phagocytosis, are attenuated during hibernation. Discovery proteomics identified 132 differentially regulated neutrophil proteins under hibernation suggesting a causal link between altered protein expression and cell function (Figure 1A). We hypothesize that hibernation (i.e. long-term immobility) induced cellular traits in neutrophils and monocytes that regulate their thrombotic und thromboinflammatory capacity.

Figure 1. (A) Bacterial (green) phagocytosis by neutrophils (blue, DAPI). (B) Discovery proteomics identified 132 differentially regulated proteins (red dots) in neutrophils between hibernation and active period.

Aim 1: Defining and characterizing the role of longterm-immobility induced immune cell traits in vitro and in vivo in the context of thromboinflammation. Therefore, we will use our across-species approach (i.e. hibernating bears, SCI patients and bed-resting individuals) to study immune cell function by multicolor-flow cytometry, co-culture assays, state-of-the-art microscopy as well as in vivo model of thrombosis.

Aim 2: Investigating the role of long-term immobility-induced immune cell traits in patients with venous thromboembolic disease and myocardial infarction.

References