Principle Investigator

Scientific interest within the context of the graduate college

This project aligns with Re-Thinking Health by investigating homeostatic immune control of a common virus, the John Cunningham virus (JCV), by deciphering the identity and function of rare populations of protective T-cells in preventing JCV reactivation and progressive multifocal leukoencephalopathy (PML). Up to 80-90% of all adults are infected with JCV, but the infection rarely causes disease. In contrast, individuals with compromised T cell immunity are at risk of PML, with high mortality and limited treatment options. Resolving antigenic targets and functional identity of protective T-cell responses, will enable design of novel vaccines and enable a more precise risk prediction in at-risk populations, such as patients under immune suppressive therapies. The program’s focus on health as an active regulated biological state, together with its emphasis on prevention-oriented translational research, provides an ideal framework to investigate adaptive immune mechanisms underlying latent viral control and maintenance of organismal health.

Project description

Human polyomaviruses are a family of DNA viruses that primarily establish persistent asymptomatic infections but can reactivate into virulent archetypes under conditions of immunosuppression. One such example is JCV, which exhibits tropism for oligodendrocytes in the central nervous system (CNS) upon reactivation, leading to the development of PML. PML is a rare but often fatal demyelinating disease of the CNS, with no licensed treatment options available to-date. Interventions including immune checkpoint inhibitors have yielded heterogenous response rates, and restoration of JCV-specific T-cell immunity currently remains the single most effective strategy to control CNS viral load and PML. Yet, this is not always possible, leaving a high unmet clinical need to better treat this debilitating disease.

While recent advances have opened promising opportunities for treatment of active PML, such as immune checkpoint blockade or adoptive T-cell transfer, our basic understanding of antigen specificity and functional capacity of protective T-cell immunity against PML remains surprisingly limited. This gap is largely attributable to technical challenges associated with detecting JCV-specific T-cells, which are present at exceptionally low frequencies in the periphery of healthy individuals, despite effective immune control. This hinders effective design of predictive assays for individuals under immunosuppression and also represents a barrier to identify protective T cells, TCRs, and their cognate epitopes profile; thus, posing a major roadblock in preventing and treating JCV reactivation and disease (PML).

To address this, we have previously i) developed a highly sensitive assay to detect rare JCV-specific CD4 T-cells from donor and patient derived peripheral blood, and ii) established a platform to isolate antigen-specific T-cell clones, and analyze their TCR and the cognate peptide epitopes, and extract transcriptomic signatures at single cell resolution. We are currently validating a sizeable set of CD4 TCR and peptide pairs. In the proposed project, we aim to extend this methodology to identify CD8 T cells, their TCRs, and MHC-I restricted epitopes. This will not only yield critical insights into JCV immune control, but it will provide a blueprint to study T cell dependent immune control to chronic viruses and build valuable datasets for machine learning based prediction of TCR – peptide pairing.

Aim 1. Adapt and validate T cell detection assay of JCV-specific CD8 T-cells.

Aim 2. Uncover JCV-specific CD8 T-cell clones, TCRs, their transcriptional profile and their cognate epitopes.

Aim 3. Validate the identified viral epitopes and TCRs by re-expression of TCRs.

Taken together, the proposed project will shed new light on immune control of JCV. Uncovered T-cell epitopes and TCRs present an attractive basis for the development of patient-specific cellular precision therapies.

Application details

References

Prinicipal Investigator

Scientific interest within the context of the graduate college

Scientific interest centers on the molecular mechanisms that preserve health and resilience with focus on microbiome-host crosstalk, especially microbiome-derived hydrogen sulfide, protein persulfidation, and systemic energy metabolism in organ function. This aligns with the goals of Re-Thinking Health, which aims to define health-preserving pathways and translate them into prevention-oriented strategies.

Project description

Our research group is investigating the role of the microbiome in kidney and cardiovascular diseases.^1,2 Interestingly, patients with kidney disease face a substantial cardiovascular risk.³ An altered microbiome may contribute to an increased cardiovascular risk in the presence of existing kidney dysfunction.⁴ However, the exact mechanisms remain poorly understood, leading to a lack of therapeutic options. We are particularly interested in the molecules that mediate the interaction between the microbiome and host cells.^5-9

Hydrogen sulfide (H₂S) is best known as the gas that smells like rotten eggs, yet in the body it also acts as an important signaling molecule.¹⁰ It can modify proteins through persulfidation, influence mitochondrial energy production, and shape inflammatory responses. These functions are highly relevant to common chronic diseases such as heart failure and chronic kidney disease. The intestinal microbiota is a major source and regulator of H₂S, but we still do not know how microbiome-derived H₂S reaches distant organs, how it changes tissue function, or whether this pathway can be harnessed therapeutically.

Our hypothesis is that microbiome-derived H₂S supports systemic resilience by maintaining organ-specific persulfidation patterns and efficient cellular energy metabolism. Loss or dysregulation of this pathway increases susceptibility to heart, kidney, and immune dysfunction, particularly under stress. This proposal aims to define the mechanistic link between gut microbial H₂S production and remote organ function in a biologically innovative and clinically relevant manner.

Aim 1: Define how the microbiome regulates systemic H₂S. We will identify how the microbiome contributes to systemic H₂S homeostasis. Using germ-free and colonized mice, together with human microbiome samples, we will profile microbial pathways involved in H₂S production by shotgun sequencing and targeted qPCR. We will then quantify H₂S levels, key precursors, and protein persulfidation across the gut and distant organs to test whether a measurable gut-to-organ gradient exists. In parallel, we will assess host H₂S -producing and H₂S -metabolizing enzymes to determine whether the microbiome also alters host H₂S synthesis or breakdown.

Aim 2: Test how microbial H₂S shapes remote organ resilience. We will test how microbiome-derived H₂S affects the heart, kidney, immune system, and intestine. We propose that reduced microbial H₂S alters the persulfidome and weakens mitochondrial function in energy-demanding tissues. Germ-free and colonized mice will be compared at baseline and in a stress model of heart failure with kidney injury. Organ phenotypes will be linked to bioenergetics, inflammation, and tissue persulfidation, while rescue experiments with an oral H₂S donor will test causality.

Aim 3: To strengthen translational relevance. We will assess the H₂S-producing capacity of the microbiota in patients with end-stage kidney disease compared to healthy controls to determine whether modulation of microbial H₂S production could serve as a novel strategy to enhance resilience and limit organ damage.

Application details

References

1. Wilck N, Matus MG, Kearney SM, Olesen SW, Forslund K, Bartolomaeus H, Haase S, Mähler A, Balogh A, Markó L, Vvedenskaya O, Kleiner FH, Tsvetkov D, Klug L, Costea PI, Sunagawa S, Maier L, Rakova N, Schatz V, Neubert P, Frätzer C, Krannich A, Gollasch M, Grohme DA, Côrte-Real BF, Gerlach RG, Basic M, Typas A, Wu C, Titze JM, Jantsch J, Boschmann M, Dechend R, Kleinewietfeld M, Kempa S, Bork P, Linker RA, Alm EJ, Müller DN. Salt-responsive gut commensal modulates T_H17 axis and disease. Nature. 2017; 551(7682): 585-589.
2. Holle J, Bartolomaeus H, Löber U, Behrens F, Bartolomaeus TUP, Anandakumar H, Wimmer MI, Vu DL, Kuhring M, Brüning U, Maifeld A, Geisberger S, Kempa S, Schumacher F, Kleuser B, Bufler P, Querfeld U, Kitschke S, Engler D, Kuhrt LD, Drechsel O, Eckardt KU, Forslund SK, Thürmer A, McParland V, Kirwan JA, Wilck N, Müller D. Inflammation in children with CKD linked to gut dysbiosis and metabolite imbalance. J Am Soc Nephrol. 2022; 33(12): 2259-2275.
3. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. New Engl J Med. 2004; 351(13): 1296-1305.
4. Bloom PP, Garrett WS, Penniston KL, Winkler MH, Hazen SL, Agudelo J, Suryavanshi M, Babiker A, Dodd D, Fischbach MA, Huang KC, Huttenhower C, Joe B, Kalantar-Zadeh K, Knight R, Miller AW, Rabb H, Srivastava A, Tang WHW, Turnbaugh PJ, Walker AW, Wilck N, Xu J, Yang T, Himmelfarb J, Redinbo MR, Wu GD, Woodworth MH, Ackerman AL, Winter S, Rinschen MM, Hassan HA, Biruete A, Anderson AH, Pluznick JL. Microbiota and kidney disease: the road ahead. Nat Rev Nephrol. 2025; 21(10): 702-716.
5. Yarritu A, Anders W, Thiele A, Potapenko O, Schumacher F, Szijártó IA, Matz-Rauch A, Versnjak J, Gebremedhin N, McParland V, Heckscher S, Kamboj S, Trimarchi G, Anandakumar H, Fuckert F, Hoffmann C, Hassan SA, Bonnekoh PM, Wimmer MI, Behrens F, Voelkl J, Kramann R, Kintscher U, Kuehne T, Kleuser B, Zernecke A, Oefner PJ, Gronwald W, Dettmer K, Eckardt KU, Müller DN, Kelm M, Holle J, Bartolomaeus H, Wilck N. Kidney disease reprograms microbiome-host signaling to promote heart failure. bioRxiv. 2026. 2026.2001.2010.698142.
6. Wimmer MI, Reichel M, Thiele A, Yarritu A, Matz-Rauch A, Anandakumar H, Götz LH, Lesker TR, Potapenko O, Gebremedhin N, Anders W, Liévano Contreras SV, Wang R, Nonn O, Schiattarella GG, Schaefer F, Holle J, Strowig T, Zernecke A, Eckardt KU, Knauf F, Wilck N, Bartolomaeus H. Interleukin-17A mediates cardiorenal injury in oxalate nephropathy. bioRxiv. 2025. 2025.2011.2017.687153.
7. Wimmer MI, Bartolomaeus H, Anandakumar H, Chen CY, Vecera V, Kedziora S, Kamboj S, Schumacher F, Pals S, Rauch A, Meisel J, Potapenko O, Yarritu A, Bartolomaeus TUP, Samaan M, Thiele A, Stürzbecher L, Geisberger SY, Kleuser B, Oefner PJ, Haase N, Löber U, Gronwald W, Forslund-Startceva SK, Müller DN, Wilck N. Metformin modulates microbiota and improves blood pressure and cardiac remodeling in a rat model of hypertension. Acta Physiol (Oxf). 2024; 240(11): e14226.
8. Bartolomaeus H, Balogh A, Yakoub M, Homann S, Markó L, Höges S, Tsvetkov D, Krannich A, Wundersitz S, Avery EG, Haase N, Kräker K, Hering L, Maase M, Kusche-Vihrog K, Grandoch M, Fielitz J, Kempa S, Gollasch M, Zhumadilov Z, Kozhakhmetov S, Kushugulova A, Eckardt KU, Dechend R, Rump LC, Forslund SK, Müller DN, Stegbauer J, Wilck N. Short-chain fatty acid propionate protects from hypertensive cardiovascular damage. Circulation. 2019; 139(11): 1407-1421.
9. Bartolomaeus H, Avery EG, Bartolomaeus TUP, Kozhakhmetov S, Zhumadilov Z, Muller DN, Wilck N, Kushugulova A, Forslund SK. Blood pressure changes correlate with short-chain fatty acid production potential shifts under a synbiotic intervention. Cardiovasc Res. 2020; 116(7): 1252-1253.
10. Filipovic MR, Zivanovic J, Alvarez B, Banerjee R. Chemical Biology of H₂S Signaling through Persulfidation. Chem Rev. 2018; 118(3): 1253-1337.

Principal Investigator

Scientific interest within the context of the graduate college

Rheumatoid Arthritis (RA) represent a chronic and prevalent autoimmune disease characterized by inflammation and progredient joint destruction. Both anti-citrullinated protein antibodies (ACPA) and autoreactive ACPA-producing B cells play a key role in disease pathogenesis. However, the exact mechanisms leading to disease onset remain poorly understood. Notably, ACPA are not only present in RA patients, but can be also detected in a certain fraction of healthy individuals where they are indicating an increased risk of developing RA in the future. The proposed study thus aims to investigate the role of autoreactive ACPA-producing B cells in ACPA-positive healthy individuals and to understand the molecular mechanisms that eventually lead to the onset of inflammatory disease. By focusing on autoreactive B cells, we aim to uncover novel molecular insights into the pathogenesis of RA and identify potential therapeutic targets to prevent disease progression.

Project description

We have collected a cohort of ACPA-positive healthy individuals as well as of ACPA-positive RA patents. The project aims to perform a molecular phenotyping of B cells in these patients where we seek to understand the behavior, characteristics and differences of autoreactive cells in ACPA-positive healthy individuals and RA patients. We plan to apply cutting edge technology including bar-coded tetramers, mass cytometry and single cell sequencing to characterize autoreactive ACPA-producing B cells. The overall aim is to understand the mechanisms that lead to onset of disease.

Aim 1: Examine the phenotypic and functional characteristics of autoreactive B cells in ACPA-positive individuals and RA patients using mass cytometry. We will use mass cytometry to perform a deep phenotyping of PBMCs of ACPA-positive individuals and RA patients where we will focus on activation markers and signaling pathway activity in response to ex vivo stimulation.

Aim 2: Examine the phenotypic and functional characteristics of autoreactive B cells in ACPA-positive individuals and RA patients using scRNAseq. Using scRNAseq and bar-coded tetramers, we will focus on the molecular phenotype of ACPA-producing autoreactive B cells in ACPA-positive individuals and RA patients.

Aim 3: Clinical Correlation. Access to clinical datasets will allow assessment of the correlation between B cell characteristics and clinical outcomes in ACPA-positive individuals.

Application details

References

Principle Investigator

Scientific interest within the context of the graduate college

The epithelium of the gastrointestinal tract is a single cell layer organized into villi and crypts, which separates microbiota and host. Stem cells in the crypts constantly generate epithelial progenitors that differentiate and migrate toward the villus tip, where they are shed into the lumen. While some of these processes are epithelial-intrinsic, coordinated interactions with underlying stromal cells are essential for tissue morphogenesis, stem cell maintenance, epithelial differentiation and spatial zonation along the crypt–villus axis. Importantly, disruption of this communication not only impairs local tissue integrity but also drives systemic inflammation, metabolic dysfunction, and cancer risk. Understanding these interactions will provide key insights into the mechanisms linking tissue-specific processes to overall organismal health and disease.

In our lab, we specifically investigate how intestinal stromal and epithelial cells communicate to maintain tissue homeostasis. Recently, we identified the transcription factor c-Maf as a key regulator of enterocyte differentiation controlled by stromal Noggin/BMP signals, linking spatial patterning along the villus axis to nutrient absorption and epithelial function. Disruption of this pathway impairs enterocyte maturation and alters immune and microbial homeostasis. Our current work focuses on the molecular pathways, cellular interactions, and spatial organization that coordinate stromal–epithelial crosstalk within the gut microenvironment. By integrating cell biology, genomics, and advanced imaging approaches, we aim to define how these interactions are perturbed in intestinal disease and to identify strategies to restore intestinal function.

Project description

In the healthy gut, the delicate balance between intestinal epithelial cell (IEC) proliferation, differentiation and apoptosis maintains barrier integrity, however, chronic inflammation disrupts this homeostasis. While stromal cells are known to regulate IEC fate via niche-derived signals, the specific molecular mediators governing these interactions remain incompletely defined. Our preliminary work identifies the opioid growth factor (OGF) and its receptor (OGFR) pathway as a previously unrecognized signalling axis between intestinal stromal cells and IECs, that is markedly upregulated during gut inflammation. Based on these findings, this project aims to comprehensively characterize this crosstalk, its regulation and functional relevance for IEC and gut homeostasis and inflammation.

To define the signals that regulate intestinal OGF/OGFR expression. While IECs are known to express OGFR and specific stromal cell populations produce OGF, the upstream signals controlling their expression remain poorly understood. Based on preliminary data, we hypothesize that homeostatic cues, including microbial- and immune cell-derived signals, regulate OGF/OGFR expression under steady-state conditions. Furthermore, our findings of increased OGF/OGFR expression during intestinal inflammation suggest that pro-inflammatory cytokines also contribute to their regulation. The objective of this project is to systematically identify and characterize the signals governing OGF/OGFR expression in the intestine.

Aim 1: Which signals regulate OGFR expression in intestinal IECs? 3D intestinal organoids (“gut in a dish”) are generated from primary mouse intestinal epithelial cells (IECs) using cell culture techniques. Cytokine stimulation experiments are then performed with these 3D intestinal organoids. Following stimulation, OGFR expression in the organoids is analyzed by quantitative PCR (qPCR). In addition, primary mouse IECs are isolated from different transgenic mouse strains (e.g., microbiota-deficient or immunodeficient mice) using fluorescence-activated cell sorting (FACS). Finally, endogenous OGFR expression in the FACS-sorted IECs is measured by qPCR.

Aim 2: Which signals regulate OGF expression in intestinal stromal cells? Primary mouse intestinal stromal cells are isolated using fluorescence-activated cell sorting (FACS). These cells are then cultured under appropriate cell culture conditions. Cytokine stimulation experiments are subsequently performed with the mouse intestinal stromal cells. Following stimulation, OGF expression in the stromal cells is analyzed by quantitative PCR (qPCR). In addition, gut tissue samples are prepared for immunofluorescence (IF) microscopy, including embedding, sectioning, and mounting. Finally, endogenous OGF expression is analyzed by IF microscopy in OGF reporter mice.

This project will combine different in vitro and in vivo approaches to define the signals that regulate OGFR and OGF expression in intestinal IECs and stromal cells, respectively. Key techniques will include isolation of primary mouse intestinal IECs and stromal cells, cell culture, 3D intestinal organoids, flow cytometry and flow cytometry-mediated cell sorting, tissue embedding, cryosectioning, staining, immunofluorescence microscopy and qPCR.

Application details

References

Principal Investigator

Scientific interest within the context of the graduate college

Our lab investigates fundamental mechanisms of communication between the nervous and immune systems, two major sensory networks that continuously monitor tissue integrity and initiate protective responses upon disruption. While the immune system’s role in maintaining barrier function is increasingly understood, the contribution of neuronal signals to immune regulation remains largely unexplored. Recent work from the lab has revealed how neuropeptides such as neuromedin U and neurotransmitters like norepinephrine modulate innate lymphoid cells (ILCs), key tissue-resident immune cells at mucosal barriers. By integrating advanced genetic tools with state-of-the-art methods from immunology, neuroscience, and genomics, the lab aims to dissect the cellular and molecular circuits of neuro-immune interaction.

Project description

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

Application details

References

Principal Investigator

Scientific interest within the context of the graduate college

Pulmonary hypertension is a common cardiopulmonary disease that is characterized by extensive remodeling of the pulmonary vascular tree and a poor prognosis due to ultimate right heart failure. The underlying progressive lung vascular remodeling involves two distinct but interconnected pathologies: distal capillary loss (vascular rarefaction) and proliferative remodeling of precapillary resistance vessels. Current therapeutic approaches rely on vasodilatory drugs and do not address the underlying causes of vascular remodeling which are poorly understood. Yet, deeper insight into the underlying pathophysiological processes – which forms the basis for the development of causal rather than symptomatic therapies – is hampered by limited access to human biosamples, the restriction to post-mortem end-point analyses in animal models, and the complexity of the multicellular processes driving vascular remodeling which cannot be recapitulated in traditional cell culture. To overcome this gap in methodologies, knowledge and ultimately therapy, we have engaged in a collaboration with a Suisse bioengineering lab to develop in-vitro microvasculature-on-chip and artery-on-chip models that allow for the first time to track changes of the vascular system and the dynamics of individual cell types in an unprecedented temporally and spatially resolved context.¹ These models – in combination with advanced imaging modalities, state-of-the-art metabolic assays and functional read-outs – are now ready to use and open up unprecedented avenues for the discovery of new mechanisms of health and disease and the development of new therapeutic targets.

Project description

Pulmonary hypertension is an ultimately fatal disease characterized by an increase in pulmonary vascular resistance that results from a combination of capillary rarefaction and inward remodeling of pulmonary arteri(ol)es.² Recently, pericytes have been implicated in both of these events: 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 closely communicate with endothelial cells promoting the stabilization of endothelial cells and the maintenance of vascular barrier function. In vitro, pericytes can even “donate”, i.e. transfer mitochondria to endothelial cells via so-called tunnelling nanotubes (TNTs).³

In pulmonary hypertension, however, pericytes detach from capillary networks, possibly contributing to their disintegration.⁴ In parallel, pericytes emerge at the level of pulmonary arteries where they integrate into the muscular layer of the vascular wall likely contributing to medial thickening.⁵ In previous work, we have shown i) that pericytes form TNTs with lung microvascular endothelial cells in intact lung capillary networks, ii) that these interaction sites show abundant expression of connexin 43, a gap junctional molecule known to coordinate mitochondrial transfer between cells,⁶ and iii) that pulmonary artery endothelial cells in pulmonary hypertension undergo a metabolic switch from oxidative phosphorylation to glycolysis (a phenomenon known from cancer cells as “Warburg-effect” that allows cancer cells to hyperproliferate). Based on these observations and published data demonstrating a similar metabolic switch in pericytes in pulmonary hypertension,⁷ we propose a paradigm-shifting metabolic concept for the co-occurrence of capillary loss and arteriolar remodeling in pulmonary hypertension that positions the pericyte in the center of the pathophysiology: In healthy homeostasis, pericytes closely interact with microvascular endothelial cells, supporting their oxidative phosphorylation by transferring mitochondria in a connexing 43-dependent manner to sustain capillary homeostasis. In pulmonary hypertension, pericytes undergo metabolic reprogramming toward increased glycolysis, resulting in their detachment from capillaries and migration to upstream resistance vessels where they integrate into the vascular wall, transferring glycolytic mitochondria to pulmonary artery smooth muscle cells thus in turn promoting their hyperproliferation and subsequent arteriolar remodeling while capillaries now disassemble in the absence of mitochondrial support from pericytes.

Our microvasculature-on-chip and artery-on-chip models in conjunction with classic co-culture systems are uniquely positioned to address this hypothesis via the following research aims:

Aim 1. To map mitochondrial transfer dynamics in lung capillary networks in health and pulmonary hypertension, we will use multi-color confocal and multi-photon imaging to track in real-time the interaction between pericytes and endothelial cells (identified by different fluorescent markers) and the transfer of fluorescently labelled mitochondria from one to another via TNTs. Experiments will be run in vascular networks formed by primary vascular cells from healthy donors and those from patients with pulmonary hypertension with the expectation that in the latter scenario, pericytes will detach from the capillaries and stop transferring mitochondria to lung microvascular endothelial cells, resulting in capillary disintegration and network rarefaction. Using dedicated assays for mitochondrial metabolism (Seahorse stress assays, Oroboros assays, metabolomic analyses by GC-MS) we will analyze the effects of this mitochondrial transfer (or lack thereof) on endothelial metabolism and function (in terms of proliferation versus apoptosis). The proposed regulatory role of connexin 43 will be tested using pharmacological inhibitors or enhancers of connexin 43-mediated gap junctional communication.

Aim 2. Using a newly engineered arteriole-on-a-chip platform, we will next probe whether pericytes with a pharmacologically or genetically induced glycolytic shift preferentially integrate into the arterial wall and (instead of promoting oxidative phosphorylation in lung capillaries) now start to donate glycolytic mitochondria to pulmonary artery smooth muscle cells, driving their proliferation. An analogous set of imaging, metabol(om)ic and functional assays will be applied as for Aim 1.

Aim 3. Finally, we will utilize a presently custom-designed platform combining a capillary network with a feeding arteriole on a single organ-on-a-chip to track the complete cascade of events from initial capillary homeostasis with quiescent pericytes to pericyte glycolytic switching, detachment and migration to the upstream arteriole where they ultimately trigger vascular remodeling by donating “injurious”, i.e. glycolytic mitochondria to smooth muscle cells.

The results of the proposed are expected to radically reshape our understanding of pulmonary hypertension, positioning changes in pericyte metabolism at the very center of the pathophysiology and identifying strategies to restore pericyte quiescence and homeostatic metabolism as a promising new avenue for the treatment of a presently uncurable disease.

Application details

References

Principal Investigator

Scientific interest within the context of the graduate college

Our group 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 (Charité 3R), to investigating the role of NF-κB in cellular senescence in the gut (DFG) or its role in metabolism.

Project description

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.

Application details

References