Principle Investigator

Scientific interest within the context of the graduate college:

A hallmark of adaptive immunity is the clonal selection and expansion of cells with somatically diversified receptors and their long-term maintenance as memory cells. The innate immune system, in contrast, is wired to rapidly respond to pathogens via a broad set of germline-encoded receptors, acquiring epigenetic imprinting at the population level. One exception to this paradigm are Natural Killer (NK) cells, which can undergo specific expansion in response to murine and human Cytomegalovirus (CMV), raising conceptual parallels to adaptive immunity.^1-3 In our more recent work, we have applied a single-cell multi-omic approach and leverage lineage tracing of human NK cells using mitochondrial mutations as endogenous barcodes⁴ to study clonality within the innate immune system and track NK cell memory to HCMV infection ex vivo. We have shown for the first time that HCMV induces the drastic expansion and differentiation of NK cell clones expressing the HCMV-specific receptor NKG2C,⁵ supporting a novel immunological paradigm of innate clonality.⁶

Project description:

Introduction: Based on this novel data, in this project we aim to investigate the dynamics of in vivo generation and maintenance of NK cell clonal expansions in response to primary HCMV infection.

To this aim, we will study NK cells isolated from HCMV- patients undergoing kidney transplantation from HCMV+ donors. These transplanted patients have a high risk of HCMV infection, which is regularly monitored in the clinic by measuring HCMV DNAemia and/or serology. Therefore this setting uniquely enables us to study the dynamics of primary HCMV infection, which is generally asymptomatic in healthy individuals. The cooperation with the nephrology clinic is already established, the ethical approval is already in place and prospective patient sample collection has already started. NK cells will be isolated from patient blood over time, i.e. between day 10 and day 30 after transplantation, during HCMV reactivation (typically around day 30) and at later time points (day 60-120). DOGMAseq, i.e. single-cell transcriptional, epigenetic, proteomic and lineage tracing analysis of NK cells will be performed, as previously published.^5,7 The computational analysis of this large set of multiomic data will be performed using already established pipeline. Moreover, clinical and laboratory parameters along with sequencing of HCMV relevant proteins will be assessed. This project will uniquely provide an overview on the dynamics of innate clonality generation and maintenance in vivo and will enable the candidate to become familiar with important immunological and molecular biology technologies, such as flow cytometry and single-cell multiomc analysis as well as with bioinformatic analysis of data sets.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our research project entitled “Identification of a gut microbiota signature in patients with drug-resistant epilepsy upon ketogenic diet treatment” aims to characterize the composition of the gut microbiota of patients affected by drug-resistant epilepsy undergoing ketogenic diet treatment, with the ultimate goal of developing probiotics as therapeutic tool. This research is characterized by combining both the clinical analysis of patients with epilepsy (encephalogram, NMRI, epigenetic changes and questionnaires on lifestyle and quality of life) and the molecular and cellular analysis of patients’ blood and stool. This project stands out for its strong clinical and experimental approach in close collaboration with the Clinic for Paediatrics and Neurology, Charité. The successful candidate will benefit from a training in which he/she will be able to put into practice his/her knowledge of the medical field and learn concepts, techniques and strategies of experimental research in the laboratory, paving the way to better understand how we could modulate the course of epilepsy via microbiota-diet modulation.

In this research project, the successful candidate will be able to:

• manage human sampling collection for research purposes.
• learn about the ketogenic diet as a therapy in the clinics
• learn about patient’s group stratification according to the different profiles in response to the treatment
• prepare and analyze questionnaires to track dietary habits, lifestyle habits, and quality of life
• extract and quantify genomic DNA from human stools
• get knowledge in microbial composition bioinformatic analysis

Project description:

Epidemiology and social impact of epilepsy and drug-resistant epilepsy (DRE): Epilepsy is one of the most common neurological disorders, affecting more than 50 million people worldwide and 6 million people in Europe (Singh & Sander, 2020; Sirven, 2015). It affects people of all ages and is characterized by epileptic seizures (Berg et al., 1999; Fiest et al., 2017; Guerrini, 2006; Sirven, 2015). Uncontrolled seizures can lead to developmental delay, cognitive deficits, memory and learning difficulties. They can also cause sudden unexpected death in people with epilepsy (SUDEP) (Sperling, 2004). Current treatments include anti-seizure medicines (ASM), diet, epilepsy surgery and stimulation devices. ASM can control seizures in about two-thirds of people with epilepsy. However, one-third of patients with epilepsy cannot be controlled by two or more correctly selected and dosed ASM, what is called drug-resistant epilepsy (DRE) (Kwan et al., 2009, 2011; Picot et al., 2008; Sultana et al., 2021).

The microbiota-gut-brain axis: a modulator for epileptogenesis: The causes of epilepsy include structural, genetic, infectious, metabolic and immune factors (Guerri et al., 2020; Matin et al., 2015; Vezzani et al., 2016; Oliver et al., 2023; Rho & Boison, 2022). The gut-brain axis (GBA) has also been implicated in this respect (Iannone et al., 2019). Gut microbiota produces a variety of compounds that can affect brain function, such as neurotransmitters and metabolites, like short-chain fatty acids (SCFAs) (Chen et al., 2021; Dalile et al., 2019; Silva et al., 2020), some of which have shown anticonvulsant effects in rodents (De Caro et al., 2019; Mu et al., 2022; Olson et al., 2018). In patients, studies have confirmed that bacterial-based approaches, such as probiotics or microbiota-modifying treatments, can reduce seizure frequency. Indeed, the pilot usage of a mixture of probiotics (eight bacterial subspecies of Lactobacillus, Bacteroides and Streptococcus) reduced seizure frequency and improved the quality of life in patients with DRE (Gómez-Eguílaz et al., 2018). Similarly, fecal microbiota transplantation (FMT) for the treatment of Crohn’s disease (CD) symptoms ameliorated seizures frequency in a 22-year-old woman with a 17-year history of epilepsy, who stopped taking ASM after the FMT (He et al., 2017). Despite these promising results, the mechanisms by which the microbiota may affect the development of seizures in patients are completely unclear.

Ketogenic diet in the management of DRE: Alternative treatments for DRE include dietary treatments such as the ketogenic diet (KD) (Hemingway et al., 2001; Lyons et al., 2020). KD is a diet based on a high fat intake accompanied by moderate protein, and very low or no carbohydrate content, usually in a 3:1 or 4:1 ratio of caloric intake from lipids to both protein and carbohydrate together, and with a normal total energy intake. Restricting carbohydrate intake forces the metabolism to replace the preferred energy source, glucose, with fatty acids, which are oxidized in the liver to form ketone bodies. Although the classical KD has been used in epilepsy for many years and its efficacy in reducing seizures has been confirmed in several studies, especially when introduced in young age (Martin-McGill et al., 2020), the mechanism behind its action is not well understood in humans. In particular, it is not known why this dietary intervention has an anti-seizure effect in a proportion of the patients, while in others there is poor or no effect (Martin-McGill et al., 2020) or even worsening of seizure frequency or appearance of adverse effects (Cai et al., 2017; Newmaster et al., 2022; Yan et al., 2018). Although the exact mechanism is not yet known, several research studies in both mice and human have shown a link between the gut microbiota and the effects of the KD, topic that we recently discussed (Díaz-Marugan et al., 2024).

Hypothesis: We hypothesize that the success of KD treatment in patients with drug-resistant epilepsy is related to the composition and function of the patient’s gut microbiota, so it would be possible to use the bacterial characteristics as biomarker to assess KD efficacy and as a probiotic tool to improve the response in those patients with a low KD-responsive profile. To test our hypothesis, our objectives are:

Aim 1: Identification of a KD-specific bacterial signature.

• To identify differences in bacterial composition and function in a large cohort of patients who respond or do not respond to KD.
• To track bacterial changes throughout the KD intervention and correlate them with seizure improvement or worsening.
• To analyze the duration of the effect of the ketogenic diet on both seizure improvement and gut microbiota changes even after the end of the intervention.

Aim 2: Isolation of bacteria associated with amelioration of seizure upon KD introduction.

• To isolate the most representative bacteria from KD responder patients.

Aim 3: Preclinical Model.

• To dissect the mechanisms of action of specific bacteria isolated from KD responders in gnotobiotic preclinical mouse models of epilepsy.

The successful candidate for this fellowship will focus on addressing aim 1. However, depending on the progress of the project (Table 1) it will be possible to start the experiments related to aim 2 (isolation of potential beneficial bacteria associated with a positive outcome of KD). This will be performed in the anaerobic chamber of our lab, through the cultivation of the bacteria from the human feces’ samples and in collaboration with Dr. Thomas Clavel (Uniklinik RWTH Aachen, Germany) using methods for single-cell dispensing of bacteria from human samples, as reported (Afrizal et al., 2022).

Table 1. Timeline. Isolation of bacteria will be possible depending on the development of the aim 1 of the research project.

Abbreviations: ASM: anti-seizure medicines; DRE: drug-resistant epilepsy; FatsQ: fats-type questionnaire; FFQ: food frequency questionnaire; KD: ketogenic diet; LifeSQ: lifestyle questionnaire; QOLIE 31: quality of life in epilepsy questionnaire

References

Principle Investigator

Scientific interest within the context of the graduate college:

Vaccines constitute one of the most effective public health interventions and play a crucial role in the control and prevention of infectious diseases.¹ Their value to human health was recently demonstrated during the COVID-19 pandemic, when the rollout of vaccines played a pivotal role in lowering the case-fatality and thus ending the pandemic state.² The versatility of vaccines is evident not only in their widespread use for prevention of disease from infections, but they can also lower morbidity and mortality from non-communicable diseases, such as myocardial infarction. Moreover, vaccines are explored as promising immunotherapeutics against cancer. Hence, the purposeful induction of protective immunity by means of vaccination has been shown to preserve health and to prevent disease.
Given that preventive vaccines are administered to healthy individuals with the goal of mobilizing health-preserving pathways, studying their exact mechanisms of action provides insights into protective immunity and yields important information for the targeted design of safe and effective vaccines. Pre-clinical development is usually carried out in animals, such as mice, to obtain information on immunogenicity and safety. However, significant differences in the immune system of rodents and humans, including differences in the immune cell receptor repertoires and T cell polarization (e.g. TFH cells) exist that may hamper translation for some vaccine candidates and underscore the need to establish human preclinical models of vaccination.^3,4

Project description:

Introduction: Investigating the mode of action of new and old vaccines in preclinical models poses a particular challenge due to the complexity of the human immune system and restrictions in studying molecular responses in lymphoid organs after vaccination in humans. There is an increasing need for models that accommodate the genetic, immunological, and environmental diversity of humans, to complement studies in animal models^3,5 and to accelerate translation into clinical development in human systems.

Highly effective vaccines possess the capacity to generate long-lasting, protective immune memory. In some cases, such as the measles vaccine, initial vaccination offers lifelong protection with estimated antibody titre half-lives of up to 200 years.^6,7

The generation of potent protective immune responses largely occurs in micro-anatomical lymphoid structures termed germinal centers. Germinal centers form within secondary lymphoid organs and are the site of antigenic selection, clonal expansion, somatic hypermutation, affinity maturation and class switch recombination of antibodies. These processes are driven by clonal competition for antigen and cognate T cell help provided by germinal center T follicular helper cells that promote the maturation of B cells towards memory B cells and antibody secreting cells. The composition of peripheral blood-derived mononuclear cells (PBMC) differs substantially from lymphocytes in secondary lymphoid organs.⁸ Consequently, blood cells, despite the relative ease of access, are not an optimal means of mapping of vaccine responses. Secondary lymphoid organs such as tonsils are a physiological source of bona fide germinal center TFH cells and B cells and they also provide antigen presenting cells and structures conducible to more complex immune responses. Human tonsils are also readily accessible following tonsillectomy.

Several recent publications describe ex vivo tonsil organoid culture platforms, which are suitable to study human vaccine responses in vitro.^9–13 The tonsillar cell composition and their described inherent capacity to reorganize and form germinal centers make them an attractive source of cells for the investigation of vaccine responses. The objective of this project is to establish a human tonsil organoid platform based on the described publications and, in a second step, the established organoid platform is used to investigate novel vaccine candidates and to compare different vaccine platforms and assess the responses over time. These longitudinal data will then serve to model and to predict vaccine responses to individual candidates.

Aim 1: The initial aim of the study is to establish and further adapt an organoid culture platform based on previously published protocols, which have already been piloted in our laboratory.^10,13 The culture of mechanically dissociated tonsil cells in the context of suitable culture supplements and culture conditions such as high cell density and in particular, cell culture plates with ultra-low cell adhesion leads to autonomous re-aggregation of the tonsil cells into tonsil-like organoids. Upon stimulation with vaccines, this re-aggregation process is increasingly observed. Alternative culture formats to be compared include culture in round well formats and tonsil explant cultures on collagen sponges and the addition of antigen via microfluidics systems, which has been shown to improve stimulation (unpublished data, personal communication).

Primary endpoints for the establishment of the organoid system include:

• High parametric spectral flow cytometry aided characterization of tonsil cells in steady state – and upon antigen stimulation.
• The antigen-specific establishment of germinal center responses, characterized by the differentiation of B cells into plasmablasts (detection by flow cytometry: CD19+ CD27++CD38++), immunofluorescence imaging and detection of antibodies by ELISA.
• Analysis of the expansion of vaccine- antigen specific B cell responses by direct staining of B cells with fluorescence tagged antigen.

Aim 2:

• Analysis of antigen-specific T cell responses using activation induced markers (AIM assay), in particular activation-dependent expression of CD40L, CD137, TNFa and IFNy and assessment of vaccine antigen specific TFH responses (CXCR5+ICOS+PD1+).
• Comparison of different vaccine types such as adjuvanted protein vaccines, mRNA vaccines, glycoconjugate vaccines and novel semisynthetic glycoconjugate vaccines (generated in collaboration with MPI for colloids and interfaces, Prof. Dr. Peter Seeberger).
• Investigate interpersonal variability in vaccine responses and comparison of steady state phenotype and composition of tonsillar cells with the in vitro vaccine response for generation of a predictive model for vaccine responses (in collaboration with Prof. Dr. Lisa Buchauer, Infectious Diseases, Charité).

References

Principle Investigator

Scientific interest within the context of the graduate college:

Based in the Department of Gastroenterology, Infectious Diseases and Rheumatology, our main clinical and research focus is the field of Inflammatory bowel diseases (IBD). Within the treatment of IBD, therapeutic goals have evolved from a control of symptoms to mucosal healing, with deep remission (clinical remission, biomarker remission and mucosal healing) being the ultimate treatment goal.¹ Developing a molecular understanding of deep remission is one of our main interests. By investigating states of deep remission – achieved through different treatment approaches – we aim to establish a holistic understanding of disease control in IBD to find answers to the driving question: ‘How healthy is an IBD patient in deep remission?’

Project description:

Introduction: What are necessary and underlying mechanisms leading to deep remission in IBD? Why do some patients achieve this treatment while others don’t? Within this project, we aim to better understand the mechanisms responsible for achieving a sustained deep remission and want to understand how IBD patients in deep remission differ from healthy individuals, especially in terms of their mucosal microenvironment. Therefore, the mandatory prerequisite is to deeply characterize the status of deep remission. The proposed project will be associated to the clinical study ‘InFlame’ (DRKS00031203), which is already running since early 2023. We have developed a ‘mucosa phenotyping pipeline’ with high-throughput methods to analyze e.g. interstitial fluid and mucosal microbiota from biopsies, 3D histology and imaging mass cytometry as well as components of the peripheral immune system via blood samples to comprehensively phenotype IBD patients and healthy individuals. All methods are established and running in the lab. The existing study team and the ongoing recruitment process guarantee intensive support and the provision of all necessary infrastructure.

Work package 1: Identify IBD patients already recruited who are in deep remission and collect clinical information (disease course, treatments, surgical procedures etc.).

Work package 2: Analyse mucosal microbiota (from individuals identified in WP1) at different localisations of the gastrointestinal tract to build a “mucosal microbiota atlas” and finally compare it with healthy individuals.

References

Principle Investigator

Scientific interest within the context of the graduate college:

The gastrointestinal mucosa consists of epithelial cells and a variety of surrounding stromal and immune cells. These cells act in concert to fulfill the functions of the mucosa such as nutrient absorption as well as the first line of defense against environmental threats. As one of the most prevalent immune cell types in the gut tissue, macrophages maintain mucosal barrier integrity. In addition to their role in immune defense, subsets of tissue-resident macrophages have been suggested to directly support functions of the epithelium as well as the vasculature and enteric neurons.^1,2 Our research aims to understand the interplay between the epithelium, stroma, and macrophages. We would like to understand how gut resident macrophages support mucosal homeostasis and how the epithelium and stroma in turn nourish macrophages. To address this, we are now developing new organoid and assembloid models to recapitulate the cellular networks observed in vivo.

Project description:

Introduction: In the gastrointestinal mucosa, the stromal compartment has been increasingly recognized for its role in supporting epithelial proliferation and differentiation.^3,4 Concurrently, the organization of stroma is also regulated by signals derived from the neighboring epithelium.⁵ Recently, we established an in vitro mucosa unit known as colon assembloids (Figure 1).⁵ This system combines adult mouse colon organoids with resident complex stromal subpopulations in a unified structure. The assembloid system faithfully recapitulates not only the anatomical morphology and cellular composition of colon epithelial crypts but also the compartmentalization of their stromal niche.⁵ The sophisticated self-organization of the epithelium and stroma in assembloids highlights the crucial role of their crosstalk.

Nonetheless, the current assembloid system lacks immune cells, which hinders the study of the direct interplay between the immune and other compartments. Macrophages are a critical innate immune cell type in the colon. Colonic macrophages were previously believed to originate from and to be continuously replenished by blood monocytes. However, mounting evidence suggests that gut resident macrophages may partially derive from embryonic precursors and undergo self-renewal throughout adulthood.^1,6 These resident macrophages exhibit highly specialized transcriptional features, distinct localizations, and specific functions.^1,7 However, their characteristics and interactions with epithelium/stroma remain poorly investigated on a functional level due to technical challenges, which is particularly true for the human mucosa.

Therefore, we aim to further develop our assembloid models by optimizing culture conditions for colon resident macrophages and adapting the technique for human cells. Once we establish an immuno-competent assembloid system that integrates resident macrophages, we will apply this system to explore the role of tissue macrophages in mucosal homeostasis.

Aim 1: Characterize the niche factors for colonic macrophages and modify the culture condition accordingly.

Aim 2: Generate human colonic assembloids containing resident macrophages and characterize their identity and spatiotemporal organization.

Aim 3: Investigate the functions of colonic macrophages in assembloids by macrophage depletion and exposure to bacterial virulence factors.

Figure 1. Cellular organization in murine colon assembloids.⁵ Confocal microscopy images of colon assembloids on days 1 and 4 labeled with markers for proliferative cells (KI67), colonocytes (KRT20), goblet cells (MUC2), and enteroendocrine cells (SYP). Scale bars: 100 µm.

References

Principle Investigator

Scientific interest within the context of the graduate college:

We are interested in understanding the role of the macrophages that reside within the kidney, termed kidney-resident macrophages (krMΦs). Although krMΦs are fundamental for kidney homeostasis, it is unclear how they support normal renal function and renal homeostasis. We seek to unravel the underlying mechanisms of how krMΦs mediate kidney resilience at steady state and in inflammation.

Project description:

Introduction: Invasive candidiasis (i.e. candidemia and deep-seated tissue candidiasis) is the most common fungal infection among hospitalised patients in the developed world.¹ In Germany, the burden of invasive fungal infections is unknown, because invasive fungal infections do not belong to the list of reportable infectious diseases.² Yet, the limited available data highlight that yeasts of the Candida genus are the most common pathogens causing invasive fungal infections in Germany.^2,3 The incidence of invasive candidiasis has risen dramatically over the past decades.⁴  Invasive candidiasis is associated with a high mortality rate, exceeding 40%, even when patients receive antifungal therapy.¹ The current therapies for invasive candidiasis, based on the use of antifungal drugs, have low efficacy in immunocompromised and critically ill patients, and the emergence of resistance to antifungal agents is becoming a major concern.⁵ A better understanding of the mechanistic underpinnings of the disease is needed, in order to build new, more specific treatments. The innate immune response to candida (namely monocytes and neutrophils) has been extensively studied.⁶ Surprising, the role of tissue-resident macrophages has not been adequately addressed, although they are in an ideal position to detect, respond and orchestrate the immune response to invasive candidiasis. This proposal seeks to unravel the role of tissue-resident macrophages in invasive candidiasis in murine models.

Aim 1: To investigate the early events in experimental invasive candidiasis. Our results suggest that the immune response developed a few hours after infection is crucial for disease progression. In this aim, the student will use various genetically modified mouse models to characterize (by e.g. flow cytometry, qPCR) the inflammatory response to Candida early after infection, e.g. macrophage activation, neutrophil recruitment. Herein, the student will acquire expertise in flow cytometry, qPCR, and mouse genetics (e.g. fate-mapping).

Aim 2: To investigate the early events of in vivo cell-cell interactions of Candida. In this part the student will utilize confocal microscopy and intravital (live) imaging to study the in vivo cell-cell interactions of Candida within the kidney (e.g. endothelial transmigration, phagocytosis by macrophages), in order to understand better the pathogenesis of the infection. The student will acquire training in surgical skills, live imaging and confocal microscopy.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Our lab studies the role of immune cells and inflammatory processes in the liver. During homeostasis the liver plays an important role in adaptation to environmental influences as it is constantly exposed to antigens from the gastrointestinal system, and plays a critical role in maintaining a balance between tolerance to harmless antigens (eg. food proteins or commensal bacteria) and control of pathogens.¹ When this balance is disrupted (termed maladaptation) the resulting immune-mediated changes can lead to chronic liver diseases and ultimately cancer.² Infiltration and activation of immune cells play an important role during the development of liver diseases, but the exact molecular and cellular mechanisms leading to the development of liver inflammation have not been fully elucidated until now. We are exploring the inflammatory processes during acute liver failure, metabolic dysfunction-associated steatotic liver disease and steatohepatitis (MASLD/MASH), liver cirrhosis and liver cancer in order to develop new diagnostic and therapeutic strategies. Furthermore, a better understanding of how both pro- and anti-inflammatory pathways can disrupt the homeostatic processes of a healthy liver is critical for the prevention of liver diseases in the first place.

Project description:

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, affecting a staggering 30% of the general population, and its prevalence is expected to rise further with an aging and more obese world population. MASLD is characterized by persistent inflammation and subsequent liver fibrosis, which can progress to steatohepatitis, cirrhosis and cancer. Dendritic cells (DC) have been shown to accumulate in chronic liver diseases, but their exact role in liver inflammation and fibrosis is complex and incompletely understood. In the context of MASLD/MASH, specific DC subsets correlate with disease severity in patients and drive liver pathology in mouse models.³ Furthermore, it has been shown that the lipid content of DC can significantly influence their functionality, with “lipid-high” DC displaying a proinflammatory phenotype while “lipid-low” DC seem to be rather tolerogenic.⁴ In this project, we would like to study how changes in the hepatic lipidome influence infiltration, differentiation and function of DCs and how this could be targeted to prevent or reverse the development of MASLD. For this purpose, tissue samples from both patients and mice with MASLD/MASH will be analyzed with state-of-the-art single-cell technologies, such as full spectrum flow cytometry,⁵ imaging mass cytometry and mass spectrometry imaging.⁶ This will be complemented by functional in vitro and ex vivo experiments using 2D and 3D cell culture systems.

Aim 1: Characterize the relationship between the hepatic lipidome and dendritic cell subsets in MASLD. Human and murine liver samples will be analyzed by imaging to mass cytometry to determine infiltration patterns of DC subsets and mass spectrometry imaging to characterize the hepatic lipid environment(s). Correlation of the results will determine which lipid species are associated with DC infiltration and distribution in the tissue.

Aim 2: Understand the influence of lipid species on dendritic cell differentiation and maturation in vitro. In vitro culture of primary human and murine dendritic cells with lipid species identified in Aim 1 will be used to analyze their influence on DC differentiation, activation and maturation. Readouts will include full spectrum flow cytometry, cytokine measurements as well as functional assays (e.g. antigen presentation).

Aim 3: Modulate dendritic cell functionality in vivo. Human and mouse hepatic organoids will be used to test how DC function can be targeted to prevent differentiation into pathogenic phenotypes, eg. through inhibition of uptake of certain lipid species and/or reprogramming of intracellular DC lipid metabolism.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Tissue resident immune cells maintain organ health by mechanisms that are not well understood. We propose to explore the mechanisms by which ILC crosstalk with resident macrophages to maintain homeostasis in the kidney.

Project description:

Introduction: Tissue resident immune cells are present in all organs at homeostasis, however their role in maintaining organ homeostasis vs. promoting disease remains poorly understood. We have recently interrogated the role of innate lymphoid cells expressing the activating receptor NKp46 in lupus nephritis, a prototypic autoimmune disease where autoantibodies and immune complex deposition initiate a chronic inflammatory response, which in some patients is mild, while in others severe. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blocking and genetic deficiency, we showed that tissue-resident NKp46⁺ ILC are crucial signal amplifiers of disease-associated kidney macrophage dynamics and epithelial cell injury. NKp46 signaling in a distinct subset of ILC1 instructed an unconventional immune-regulatory transcriptional program. NKp46activation instructed disease-associated macrophages to adopt pro-inflammatory programs that led to proximal tubular damage and periglomerular fibrosis. While these data revealed that NKp46 activation in ILC1 constitutes a previously unrecognized, critical tissue rheostat that regulates disease-associated macrophage dynamics and amplifies macrophage-mediated epithelial tissue damage in autoimmune hosts, the role of tissue resident ILC1 at homeostasis remains elusive, and yet, we have identified frequent physical association of ILC1 with tissue resident macrophages. Here, we hypothesize that resident ILC1 are critical for the differentiation and function of tissue resident macrophages in multiple organs. Promoting healthy interactions of resident ILC1 and macrophages may thus be a valid strategy to protect from autoimmune organ damage. To address this hypothesis, we propose the following specific aims:

Aim 1: To explore the role of the activating receptors, expressed by tissue resident ILC1, in regulating ILC1 phenotype and function in various organs, such as the kidney, small intestine and the dura mater.

Aim 2: To explore the role of ILC1 in instructing tissue resident macrophage function at homeostasis and in response to type I interferons, TLR7 ligands or immune complexes.

Aim 3: To identify mechanisms by which ILC1 are activated at homeostasis and in the presence of elevated type I interferons.

References

Principle Investigator

Scientific interest within the context of the graduate college:

Chronic kidney dysfunction is a major global health concern of increasing prevalence, driving a plethora of secondary comorbidities. A mechanistic understanding of the driving factors is essential to develop preventive and therapeutic concepts.
Our research group investigates microbiome-mediated mechanisms of cardiovascular risk (Wilck et al. Nature 2017; Bartolomaeus et al.,Circulation 2019; Avery et al., Cardiovasc Res. 2023). CKD is an understudied risk factor for cardiovascular disease. In the long term, microbiome-targeting interventions could help to reduce cardiovascular risk in CKD patients.

Project description:

Introduction: Chronic kidney disease (CKD) is a main contributor to cardiovascular (CV) risk and subsequent multimorbid conditions. CKD associates with dysbiotic alterations of the gut microbiome, a resulting dysbalance of gut bacterial metabolites, an impaired intestinal barrier function and resulting chronic systemic inflammation driving CV disease. We have recently shown that intestinal barrier dysfunction and increased AhR activity may drive inflammation and cardiovascular remodeling (Holle et al., J Am Soc Nephrol. 2022; Holle et al., manuscript in preparation). The aim of the present project is to investigate human cohort material to confirm this hypothesis and to test the microbiome dependency of these changes by means of microbiota transfer into mice.

Aim 1: Analysis of biosamples from a human CKD cohort and correlation with hallmarks of cardiovascular damage. We will use the CARVIDA (CARdioVascular In Depth Assessment in Chronic Kidney Disease) cohort, a subcohort (n=290) of the German Chronic Kidney Disease (GCKD) study. The GCKD study is a prospective cohort study encompassing 5,000 CKD patients with a follow-up of up to 10 years (incl. CV outcomes). CARVIDA additionally provides unique phenotyping of the CV condition of patients enrolled within a time frame of 4 years including cardiac MRI. Dietary protocols and plasma metabolites analyzed by NMR are available. Thus, this cohort is well-suited to identify inflammatory markers associated with CV abnormalities and to explore the relationship between gut barrier dysfunction, inflammation and CVD.

We aim to assess circulating markers of intestinal barrier dysfunction (LPS, sCD14, ZO-1). The AhR-activating potential of patient sera will be analyzed using a reporter cell-based assay and qPCR-based measurement of AhR-target genes. Results will be correlated with available metabolomics data, OLINK proteome data and markers of cardiovascular damage (blood pressure, cardiac hypertrophy, vascular function, atherosclerosis).

Aim 2: Investigation of the influence of CKD candidate bacterial species on AhR and the intestinal barrier. Gnotobiotic mice, i.e. mice with known, defined gut bacteria, provide an opportunity to mechanistically study the impact of specific bacteria on the host organism. In previous microbiome analyses (Holle et al. J Am Soc Nephrol. 2022; Holle et al. manuscript in preparation), we have identified CKD-specific candidate species that we would like to investigate by colonizing germ-free mice. The analysis will focus on the intestinal barrier (immunohistology, gene expression), AhR activity (cell-based reporter assay), inflammation (flow cytometry), as well as CV target organs (heart, vasculature).

References