The role of oxalate transporters in maintaining kidney health

Prinicipal Investigator

Prof. Dr. Felix Knauf,
PD Dr. Martin Reichel
Dr. Harald Stachelscheid

Scientific interest within the context of the graduate college:

Our laboratory focuses on the mechanisms involved in maintaining oxalate homeostasis. Oxalate is a component of various foods and is absorbed via the intestine. High urinary oxalate concentrations lead to kidney stones, the second most common kidney disease after hypertension. Furthermore, we have shown that elevated blood oxalate concentrations are associated with cardiovascular disease.1 We are working translationally and recently demonstrated that oxalate uptake in the intestine can be reduced via an enzyme isolated from bacteria in patients.2

Our research group has cloned the first oxalate transporter (SLC26A6).3 SLC26A6 is expressed in different organs. The transporter is located on the apical side of epithelia and actively secretes oxalate into the intestinal lumen4 and urine.5,6 Via this transport process, the oxalate concentration in the body is kept low. If the transporter is missing, there is an increased uptake of oxalate from the intestine, and consequently the formation of kidney stones and progressive kidney damage.7

The subject of our current scientific work is the role of oxalate in the kidney and specifically its influence on the progression of renal diseases. For this purpose, we are working with pluripotent stem cells from which kidney organoids are derived as models.

Specifically, the question of our proposed thesis project is to what extent oxalate has toxic effect(s) on kidney cells. Here we have concrete preliminary data that an accumulation of oxalate has an intracellular toxic effect and that deletion of oxalate transporter SLC26A6 promotes cell death.

Project description:

WP 1: Characterization of renal organoids from pluripotent stem cells. SLC26A6 deficient human pluripotent stem cells have been generated using CRISP/Cas technology in collaboration with BIH Stem Cell Core. As a first step, you will 1) grow kidney organoids and 2) characterize the organoids using kidney cell markers.

WP 2: Examine the effect of oxalate accumulation using kidney organoids. You will be exposing human kidney organoids to oxalate in the presence and absence of the oxalate transporter SLC26A6. You will be examining the mechanism of cell death induced by oxalate by the 1) release of LDH, 2) changes in membrane integrity and 3) the activation of Casp-3 (by western blotting, immunohistochemistry of cleaved Casp-3, and measurement of cleavage activity using fluorescent substrate).

Application details

References

  1. Pfau A, Ermer T, Coca SG, […], Aronson PS, Drechsler C, Knauf F. High Oxalate Concentrations Correlate with Increased Risk for Sudden Cardiac Death in Dialysis Patients. J Am Soc Nephrol. 2021; 32:2375-2385, doi: 10.1681/ASN.2020121793.
  2. Lieske JC, Lingeman JE, Ferraro PM, […], Tosone C, Kausz AT, Knauf F. Randomized Placebo-Controlled Trial of Reloxaliase in Enteric Hyperoxaluria. N Engl J Med Evid. 2022. doi: 10.1056/EVIDoa2100053.
  3. Knauf F, Yang CL, Thomson RB, Mentone SA, Giebisch G, Aronson PS. Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells. Proc Natl Acad Sci USA. 2001; 98:9425-9430, doi: 10.1073/pnas.141241098.
  4. Neumeier LI, Thomson RB, Reichel M, Eckardt KU, Aronson PS, Knauf F. Enteric Oxalate Secretion Mediated by Slc26a6 Defends against Hyperoxalemia in Murine Models of Chronic Kidney Disease. J Am Soc Nephrol. 2020; 31:1987-1995. doi: 10.1681/ASN.2020010105.
  5. Knauf F, Ko N, Jiang Z, […], van Itallie CM, Anderson JM, Aronson PS.Net intestinal transport of oxalate reflects passive absorption and SLC26A6-mediated secretion. J Am Soc Nephrol. 2011; 22:2247-2255. doi: 10.1681/ASN.2011040433.
  6. Knauf F, Thomson RB, Heneghan JF, […], Egan ME, Alper SL, Aronson PS. Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion. J Am Soc Nephrol. 2017; 28:242-249. doi: 10.1681/ASN.2016030279.
  7. Jiang Z, Asplin JR, Evan AP, […], Nottoli TP, Binder HJ, Aronson PS. Calcium oxalate urolithiasis in mice lacking anion transporter Slc26a6. Nat Genet. 2006; 38:474-478. doi: 10.1038/ng1762.

Innate lymphoid cells, IL-22 and liver regeneration

Prinicipal 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:

Innate lymphoid cells (ILC) are tissue-resident innate lymphocytes that are involved in immunity to infections but are also deeply integrated in the regulation of tissue function. Based on our preliminary and on published data (Gronke, Nature 2019; Guendel, Immunity 2020; Diefenbach, Immunity 2020), we hypothesize that ILC regulates 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 neuron-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.

Application details

References

  1. Witkowski M, Tizian C, Ferreira-Gomes M, […], Radbruch A, Mashreghi MF, Diefenbach A. Untimely TGFβ responses in severe COVID-19 limit antiviral function of NK cells. Nature. 2021; 600:295-301. doi: 10.1038/s41586-021-04142-6.
  2. Schaupp L, Muth S, Rogell L, […], Schild H, Diefenbach A1,*, Probst HC*. Microbiota-induced tonic type I interferons instruct a poised basal state of dendritic cells. Cell. 2020; 181:1080-1096. doi: 10.1016/j.cell.2020.04.022. 1lead senior author; *equal contribution.
  3. Guendel F, Kofoed-Branzk M, Gronke K, […], Mashreghi MF, Kruglov AA, Diefenbach A. Group 3 innate lymphoid cells program a distinct subset of IL-22BP-producing dendritic cells demarcating solitary intestinal lymphoid tissues. Immunity. 2020; 53:1015-1032. doi: 10.1016/j.immuni.2020.10.012.
  4. Gronke K, Hernández PP, Zimmermann J, […], Glatt H, Triantafyllopoulou A, Diefenbach A. Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature. 2019; 566:249-253. doi: 10.1038/s41586-019-0899-7.
  5. Hernandez P, Mahlakoiv T, Yang I, […], Suerbaum S, Staeheli P, Diefenbach A. Interferon-λ and interleukin-22 cooperate for the induction of interferon-stimulated genes and control of rotavirus infection. Nat Immunol. 2015; 16:698-707. doi: 10.1038/ni.3180.

The role of DNA damage response signaling in chronic kidney disease

Scientific interest within the context of the graduate college:

Our research group studies the underlying mechanisms of chronic kidney disease (CKD). Acute and chronic kidney injury has been increasingly recognized as a global public health concern, associated with high morbidity, and mortality. Acute kidney injury is frequent, occurring in 21% of hospital admissions and leads to CKD regardless of the cause. CKD encompasses a group of heterogeneous disorders affecting renal structure and function with a prevalence of 10-15% worldwide1. During the past decades, research into kidney disease has largely focused on identifying causative insults and disease modifiers of renal injury. However, sufficient clarification about the underlying pathophysiological mechanism has not provided yet.

We hypothesize that the ability of the kidney to respond to different stress conditions contributes significantly to the maintenance of normal renal function and structure, whereas an impaired cellular stress responses promotes renal damage. Here, we focus on the so-called “DNA Damage Response” (DDR)2, which relevance for kidney diseases has been demonstrated in individuals with interstitial nephritis caused by monogenic mutations in genes encoding proteins of the DDR complex3. Interestingly, transgenic mice models with a defective DDR response show an increased susceptibility to environmental nephrotoxins, which leads to renal failure and typical histological features of CKD4. Moreover, recently published data argues that DDR functions are critical in disease progression of rare, inherited juvenile nephropathies as evidenced by accumulated DNA damage, that yields to increase apoptosis coupled with profibrotic responses5,6. This projects aims to investigate if an impaired DNA damage response contributes to the renal pathomechanisms of AKI and CKD.

Project description:

In CKD, regardless of its cause, renal fibrosis is the primary determinant of end-stage kidney disease, with no effective therapy available today. Within in scope of this project it is planned to study the response of kidney tubular cells to DNA damage, which may play a role in the pathophysiology of CKD. Therefore, we employed a human in-vitro model using patient derived renal tubular cells from urine samples, which allows investigating the biological impact of DNA damage response signaling. In a comparative approach, cultured renal tubular cells from individuals with CKD and healthy controls will be used to study the cellular response by applying different molecular techniques. In detail, it is planned to evaluate the activity of the DDR pathway targeting e.g. the accumulation of DNA damage, the cell cycle progression, the rate of apoptosis and profibrotic gene expression profiles. This project provides a new cellular model to study disease mechanisms of acute and chronic kidney injury and may offer new understandings about the pathogenesis of CKD.

Application details

References

  1. Eckardt KU, Coresh J, Devuyst O, […], Köttgen A, Levey AS, Levin A. Evolving importance of kidney disease: from subspecialty to global health burden. Lancet. 2013; 382:158-169. doi: 10.1016/S0140-6736(13)60439-0.
  2. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Mol Cell. 2010; 40:179-204. doi: 10.1016/j.molcel.2010.09.019.
  3. Zhou W, Otto EA, Cluckey A, […], Levy S, Smorgorzewska A, Hildebrandt F. FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair. Nat Genet. 2012; 44:910-915. doi: 10.1038/ng.2347.
  4. Airik R, Schueler M, Airik M, […], Mukherjee E, Sims-Lucas S, Hildebrandt F. A FANCD2/FANCI-Associated Nuclease 1-Knockout Model Develops Karyomegalic Interstitial Nephritis. J Am Soc Nephrol. 2016; 27:3552-3559. doi: 10.1681/ASN.2015101108.
  5. Chaki M, Airik R, Ghosh AK, […], Smogorzewska A, Otto EA, Hildebrand F. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling. Cell. 2012; 150:533-548. doi: 10.1016/j.cell.2012.06.028.
  6. Slaats GG, Giles RH. Are renal ciliopathies (replication) stressed out? Trends Cell Biol. 2015; 25:317-319. doi: 10.1016/j.tcb.2015.03.005.

To generate an atlas of cellular senescence in different types and severity grades of liver disease

Prinicipal Investigator

PD Dr. Cornelius Engelmann
Prof. Dr. Frank Tacke

Scientific interest within the context of the graduate college:

Cellular senescence occurs in the liver in health and disease. Senescence relates to a status of cell cycle arrest, which becomes more prevalent with increasing age and which develops as the consequence of liver disease.1 Due to subsequent changes in cell morphology and functionality senescent cells may prompt disease progression and the development of disease-related complications.1-3 Detection of senescence and deciphering of its underlying mechanisms may help identifying novel targets to develop preventive treatment strategies to halt the development of liver disease related complications such as fibrosis, inflammation, and ultimately cirrhosis.4 Therefore, this project seeks to address the following aspects:

  1. Targeting (hepatocellular) senescence may be considered as a preventive strategy to maintain health by halting disease progression to irreversible stages early in the beginning of disease development.
  2. Molecular pathways initiating hepatocellular senescence will be described and analyzed in great detail in order to lay the basis for novel interceptive therapies preventing liver diseases to establish.
  3. Ideal time points for potential interventions (senolysis) to restore health and to prevent accelerated aging as the consequence of diseases will be explored.

Project description:

Aim: Cellular senescence is a well-known consequence of diseases in general but in-depth characterization of triggers (DNA damage, Telomere shortening, etc.), pathways leading to cell cycle arrest (e.g. p16 dependent pathway, p53 dependent pathway), and subsequent cell-phenotypic changes (e.g. GATA4 driven SASP, Cyp450 expression, metabolic liver zonation) are sparse. Therefore, the main aim of this project is to decipher the characteristics of hepatocytes senescence in different types and stages of liver disease. It will serve as the basis for any type of senolytic intervention, first with respect to the type of senolytic compound [broad spectrum (e.g. BCL2 inhibitor) vs. pathway specific (e.g. p53 interacting proteins)] and second, with respect to the time point of intervention. It will be based on an in-depth analysis of human and rodent tissue samples.

Workplan: Healthy livers from different ages will be included to understand to what extent aging drives cellular senescence in the liver. The following liver diseases are relevant in Europe thus being involved in this project: 1) Paracetamol-induced liver injury 2) Drug-induced liver injury 3) Alcoholic hepatitis 4) Non-alcoholic steatohepatitis 5) Cirrhosis 6) Acute-on-chronic liver failure (ACLF). Human FFPE liver samples with different disease stages from the Pathology department Charité will be analyzed by conventional immunostaining and multiplex immunofluorescence staining with respect to DNA-damage, cell cycle arrest, autophagy, and cell death. Image analysis will be attempted to be on single-cell level including descriptive neighboring analysis. In addition, whilst regenerative mechanisms generally appreciate the proliferative activity of cells and preservation of tissue integrity, restoration of metabolic activity by alteration of liver zonation to maintain organ function is another incremental part of liver regeneration although being frequently disregarded. Therefore, expression of senescence markers will be correlated with alteration of liver zonation and metabolic characterization of hepatocytes. Mechanistic information will be obtained from already performed animal models for the above mentioned diseases as well as conditional knockout mouse lines [AhCreMdm2fl/fl (inducing senescence in hepatocytes); AhCrep53fl/fl (preventing senescence in hepatocytes)] after exposure to toxins such as lipopolysaccharides or ethanol. In addition to immunostaining analyses from paraffin-embedded tissue, frozen tissue will allow generating proteomic (mass spectrometry) and transcriptomic (mRNA sequencing) data. By using laser capture microdissection to extract proteins and RNA from areas of interest spatial information can be added to this analysis. Performing Seahorse from frozen section of liver tissue will generate more data on the metabolic/mitochondrial tissue function and will be correlated with changes of metabolic liver zonation and presence of cellular senescence in different liver diseases. All experimental techniques are well established in our lab and respective animal experiments were performed previously.

Impact: Results from this project will provide highly relevant information regarding the role of cellular senescence in homeostasis and different types of liver disease, will allow generating disease related hypothesis and may serve as the basis for later interventional study (e.g. senolytic therapies) aiming at restoring “liver health” with respect to timing of intervention and choice of senolytic compounds.

Application details

References

  1. Ferreira-Gonzalez S, Rodrigo-Torres D, Gadd VL, Forbes SJ. Cellular Senescence in Liver Disease and Regeneration. Semin Liver Dis. 2021; 41:50-66. doi: 10.1055/s-0040-1722262.
  2. Kang C, Xu Q, Martin TD, […], Yanker BA, Campisi J, Elledge SJ. The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science. 2015; 349:aaa5612. doi: 10.1126/science.aaa5612.
  3. Engelmann C,Tacke F. The Potential Role of Cellular Senescence in Non-Alcoholic Fatty Liver Disease. Int J Mol Sci. 2022; 23:652. doi: 10.3390/ijms23020652.
  4. Baar MP, Brandt RMC, Putavet DA, […], Hoeijmakers JHJ, Campisi J, de Keizer PLJ. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 2017; 169:132-147 e116, doi: 10.1016/j.cell.2017.02.031

Healthy skin homeostasis? – Does anti-cytokine treatment restore the physiological and immunological function of the skin?

Prinicipal 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 and cytokine signaling pathways.

Project description:

The skin forms the outer barrier to the environment and is in constant contact with it. Besides its function as a physical barrier, it is rich in immune cells that serve as a defense against external stimuli. In chronic inflammatory skin diseases such as psoriasis (PSO) or atopic dermatitis (AD), there is a disturbance of the cutaneous immune microenvironment. The resulting imbalance in immunological homeostasis leads to overactivation of inflammatory signaling pathways. This effect is caused and amplified in AD, HS, and PSO by specialized T helper cells and their associated cytokines (TNF-α, IL-1, IL-4, IL-5, IL-12, IL-13, IL-17, IL-22, IL-36). While AD is dominated by IL-4/IL-13 and Th2 cells, PSO is dominated by IL-17/IL-23 and Th17 cells. As a result, there has been an increased focus on blocking these cytokines and their signaling cascades either with monoclonal antibodies (biologics) or small molecules such as tyrosine kinase inhibitors (JAK inhibitors) to treat psoriasis and atopic dermatitis.

With current anti-cytokine treatments significant improvement and skin clearance can be achieved in the majority of patients in all of the indicated chronic inflammatory skin diseases. However, it remains unclear, if cleared skin under anti-cytokine treatment is comparable to healthy skin. Therefore, it is key to understand the possible differences and similarities in treated healed skin in comparison to otherwise healthy skin. This may allow to better define the duration of anti-cytokine treatment and/or risks for disease flares.

In the context of the doctoral thesis, the aim is to recruit 15 patients per disease (AD and PSO) who have been successfully treated with systemic treatments. In comparison, healthy skin samples from individuals without any inflammatory skin diseases obtained from our dermatosurgery unit will be used. The differences (immunological, physiological, barrier function) between healthy and treated/healed skin will be investigated based on gene expression of the tissue samples by whole transcriptome sequencing (WTS). As part of the immunohistochemical workup of the tissue, we will assess the immune cell infiltrate, Ki67 as a marker for (keratinocyte) proliferation and KRT16 for keratinocyte differentiation. Routine H&E staining will be used for disease characteristics and the extent of epidermal thickness. In addition, the restoration of the physiological barrier function will be assessed by the expression of filaggrin, loricrin and layer specific keratins.

References

  1. Meier K, Holstein J, Zidane M, […], Ulrich C, Ghoreschi K, Solimani F. Paradoxical lichen planus induced during anti-IL-17A treatment is immunologically different from spontaneously occurring lichen planus. J Eur Acad Dermatol Venereol. 2022; doi: 10.1111/jdv.17996. Online ahead of print.
  2. Ghoreschi, K, Balato A, Enerbäck C, Sababt R. Therapeutics targeting the IL-23 and IL-17 pathway in psoriasis. Lancet. 2021; 397:754-766. doi: 10.1016/S0140-6736(21)00184-7.
  3. Pietschke K, Holstein J, Meier K, […], Ghoreschi FC, Solimani F, Ghoreschi K. The inflammation in cutaneous lichen planus is dominated by IFN-ϒ and IL-21-A basis for therapeutic JAK1 inhibition. Exp Dermatol. 2021; 30:262-270. doi: 10.1111/exd.14226.
  4. Solimani F, Meier K, Ghoreschi K. Emerging Topical and Systemic JAK Inhibitors in Dermatology. Front Immunol. 2019; 10:2847. doi: 10.3389/fimmu.2019.02847.

Learning from outliers – Identifying determinants of kidney survival in non-progressive ADPKD

Prinicipal Investigator

Prof. Dr. Jan Halbritter
Dr. Ria Schönauer

Scientific interest within the context of the graduate college:

Not Everything Is “Genetic”, but Genes Are Involved in Everything (adapted from Kenneth M. Weiss). Our group is interested in identification and investigation of genetic, clinical, and environmental factors determining 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:

One of six patients undergoing renal transplantation has autosomal-dominant polycystic kidney disease (ADPKD) caused by heterozygous germline mutations in one of two main disease genes, namely PKD1 (encoding polycystin 1, PC1) or PKD2 (encoding polycystin 2, PC2).ADPKDis the commonest genetic disorder leading to CKD including end-stage kidney failure (ESKF).1-3 ESKF from ADPKD commonly occurs between ages 30-80 years. While PKD1-associated disease is generally more severe than PKD2-disease, exemplified by a 20-year difference in mean age at ESKF (55 versus 75 years), current genotype-phenotype correlations are still crude.4,5 For example, some patients with even identical PKD1-mutations vary dramatically in their progression. We demonstrated that additional non-diagnostic hypomorphic PKD1-germline variants, as well as variants in genes involved in proteostasis mechanistically, add to the PKD1-mutational effect.6,7 The underlying mechanisms involve posttranslational modification and endoplasmic reticular (ER)-processing of PC1. As a result, PC1 expression at the cell surface is reduced.8 Conversely, opposite mechanisms may confer renoprotection by increasing PC1 surface expression. We aim to learn from clinical outliers and hypothesize that the latter mechanisms play a role in individuals with ADPKD not depending on dialysis until their 70s and beyond. To address this, we propose the following two specific aims and work packets (WP):

Aim 1/WP1: Identification of genetic determinants associated with non-progression and kidney survival. Exome sequencing in individuals (n=20) with mildest, non-progressive ADPKD-PKD1 (non-ESKF > 70 yrs) and most severe ADPKD-PKD1 (ESKF < 40 yrs) from the Leipzig-Berlin ADPKD-cohort (n=400) via the BIH-sequencing core facility. Consecutive assessment for additional rare and common variants (single variant analysis and variant burden analysis) by use of a predefined CKD/ADPKD-candidate gene panel. Identification of differentially enriched candidate gene sets and single variants, suitable for functional analysis (WP2).

Aim 2/WP2: Validation of genetic determinants associated with non-progression and kidney survival. Overexpression of two most promising gene variants significantly associated with kidney survival and consecutive use of established cellular read-outs on RNA and protein level. Planned analyses include qRT-PCR, Western Blot, and immunofluorescence imaging (e.g. PC1 surface expression).

Application details

References

  1. Lanktree MB, Haghighi A, Guiard E, […], Harris PC, Paterson AD, Pei Y. Prevalence Estimates of Polycystic Kidney and Liver Disease by Population Sequencing. J Am Soc Nephrol. 2018; 29: 2593-2600. doi: 10.1681/ASN.2018050493.
  2. Cornec-Le Gall E, Alam A, Perrone RD. Autosomal dominant polycystic kidney disease. Lancet. 2019; 393:919-935. doi: 10.1016/S0140-6736(18)32782-X.
  3. Schönauer R, Baatz S, Nemitz-Kliemchen M, […], Neuber S, Bergmann C, Halbritter J. Matching clinical and genetic diagnoses in autosomal dominant polycystic kidney disease reveals novel phenocopies and potential candidate genes. Genet Med. 2020; 22:1374-1383. doi: 10.1038/s41436-020-0816-3.
  4. Su Q, Hu F, Ge X, […], Zhou Q, Mei C, Shi Y. Structure of the human PKD1-PKD2 complex. Science. 2018; 361: eaat9819. doi: 10.1126/science.aat9819.
  5. Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. New Engl J Med. 2011; 364:1533-1543. doi: 10.1056/NEJMra1010172.
  6. Irazabal MV, Rangel LJ, Bergstralh EJ, […],BJ, King BF, Torres VE, CRISP Investigators. Imaging classification of autosomal dominant polycystic kidney disease: a simple model for selecting patients for clinical trials. J Am Soc Nephrol. 2015; 26:160-172. doi: 10.1681/ASN.2013101138.
  7. Zhang Z, Bai H, Blumenfeld J, […], Robinson RD, Kapur S, Rennert H. Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing. J Am Soc Nephrol. 2011; 32:3114-3129. doi: 10.1681/ASN.2021050690.
  8. Durkie M, Chong J, Valluru MK, Harris P C, Ong A C M. Biallelic inheritance of hypomorphic PKD1 variants is highly prevalent in very early onset polycystic kidney disease. Genet Med. 2021; 23:689-697. doi: 10.1038/s41436-020-01026-4.

The role of the OSM pathway in maintaining intestinal homeostasis

Prinicipal Investigator

Scientific interest within the context of the graduate college:

The mammalian gastrointestinal tract contains the largest number of immune cells and harbors a large and diverse population of commensal bacteria that exist in a symbiotic relationship with the host.1,2 The gut-resident immune cells are separated from our microbial residents by a single layer of intestinal epithelial cells (IEC). The dynamic cross-talk between IEC, the intestinal microbiota, and local immune cells represents a cornerstone of intestinal homeostasis.3,4 The balance between the various immune cell populations and tonic cytokine signals play an important role in determining thresholds of tolerance and immunity in the intestine.

Project description:

We recently highlighted the relevance of Oncostatin M (OSM) in intestinal inflammation.5 OSM is a pleiotropic cytokine belonging to the interleukin 6 (IL-6) family, which influences numerous homoeostatic and pathological processes in various organs, yet its biology remains obscure.6,7 OSM receptor (OSMR) is widely expressed at both tissue (vascular system, heart, lung, adipose tissue, skin, bladder, mammary tissue, adrenal gland, and prostate) and cellular levels (endothelial, smooth muscle, fibroblast, and lung epithelial cells). In contrast, OSM is expressed in multiple hematopoietic cell types including activated monocytes/macrophages, neutrophils, dendritic cells, and T cells. We showed recently that OSMR is widely expressed by stromal and endothelial cells in the intestine; however, the role of OSM in the maintenance of intestinal homeostasis remains unknown. We hypothesize that microbiota-derived local cues induce constitutive OSM expression by gut-resident immune cells to promote intestinal homeostasis by acting on both stromal and endothelial cell compartments. This project will exploit new reporter mouse lines generated in Hegazy lab (OsmriScarlet, OsmZsgreen), gnotobiotic mice, and primary human tissue samples to explore the signals regulating OSM expression in the gut and how OSM functions as a potential tissue rheostat. The project will utilize different cellular and molecular biology techniques, including magnetic cell isolation, flow cytometry, gene expression analysis, RNA sequencing, and histology.

The following specific objectives will be addressed in this project:

  1. Characterize the influence of intestinal microbiota on the regulation of OSM pathway
  2. Identify the down-stream sensing pathways regulating OSM-OSMR axis in gut-resident immune cells
  3. Assess the relevance of the OSM-OSMR pathway in promoting intestinal homeostasis

Application details

References

  1. Belkaid Y, Hand TW. Role of the Microbiota in Immunity and Inflammation. Cell. 2014; 157:121-141. doi: 10.1016/j.cell.2014.03.011.
  2. Macpherson AJ, Slack E, Geuking MB, McCoy KD. The mucosal firewalls against commensal intestinal microbes. Semin Immunopathol. 2009; 31:145-149. doi: 10.1007/s00281-009-0174-3.
  3. Hooper LV, Littman DR, Macpherson AJ. Interactions Between the Microbiota and the Immune System. Science 2012; 336:1268-1273. doi: 10.1126/science.1223490.
  4. Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol. 2014; 14:141-153. doi: 10.1038/nri3608.
  5. West NR, Hegazy AN, Owens BMJ, […], Keshav S, Travis SPL, Powrie F. Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor-neutralizing therapy in patients with inflammatory bowel disease. Nat Med. 2017; 23:579-589. doi: 10.1038/nm.4307.
  6. Richards CD. The Enigmatic Cytokine Oncostatin M and Roles in Disease. ISRN Inflamm. 2013; 2013:512103. doi: 10.1155/2013/512103.
  7. West NR, Owens BMJ, Hegazy AN. The oncostatin M-stromal cell axis in health and disease. Scand J Immunol. 2018; 88(3):e12694. doi: 10.1111/sji.12694.

Does fasting re-program inflammatory immune cells?

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Diet is an important factor for a healthy life. For the most part of human history, the next meal was not a given. Hence, there was a strong selection pressure for adaptation to periods of no or low food consumption during our evolution. Consequently, today’s excessive calorie intake, as it is typical for diets in the western world, results in increasing occurrence of systemic inflammation and widespread diseases. In contrast, calorie restriction has been shown to improve numerous chronic diseases and to prolong the healthy lifespan. In my group, we are re-thinking health in the context of evolutionary adaptation to low food energy intake. Specifically, we focus on the identification of cellular and molecular mechanisms how reduced calorie intake maintains health, prevents and improves inflammatory diseases, and prolongs healthy life.

Project description:

The focus of my group is to understand the cellular and molecular mechanisms how reduced calorie intake regulates homeostasis and function of the immune system. Recently, we have found that fasting drastically reduces the number of circulating pro-inflammatory monocytes in the blood of humans and mice.1 Interestingly, monocytes accumulated in the bone marrow. Our preliminary data suggest that this is due to the inhibition of monocyte egress from the bone marrow as well as to recruitment from the blood circulation. This phenomenon can be observed not only for monocytes but also for specific other immune cell populations such as naïve B cells and memory T cells.2,3 Intriguingly, memory T cells that have been re-located to the bone marrow display functional modulation and improvement upon egress and re-circulation.2 Hence, our research question is: Are monocytes functionally modified in the bone marrow during fasting? To answer this question, we will establish a monocyte adoptive transfer model in mice and apply next generation sequencing to monocytes in the periphery and the bone marrow. Thus, this project offers the opportunity to the student to develop bench working skills as well as getting familiar with the computational analysis of large transcriptomic datasets.

The aim of the study is to analyze the transcriptional profile of monocytes that returned to the bone marrow from the periphery during fasting.

WP1: Establishing an adoptive monocyte transfer model into fed and fasted mice. It is crucial to the study that monocytes returning to the bone marrow during fasting can be clearly identified. Therefore, we will isolate Ly-6C+ monocytes from the spleen of CD45.1+ mice using the Miltenyi monocyte isolation kit (Fig. 1). Isolated monocytes will be transferred into CD45.2+ mice that will be fed or fasted for 16 hours via intravenous injection. After 4 hours, bone marrow, blood, spleen, and additional organs will be harvested and analyzed for transferred CD45.1+ monocytes using multicolor flow cytometry. Hence, CD45.1/CD45.2 discrimination will enable us to identify and quantify monocytes that are recruited from the blood to the bone marrow. We already obtained the approval of the responsible state office for the animal experiments.

Fig. 1. Graphical representation of study model and methodology.
BM = bone marrow.

WP2: Monocyte transcriptomics and computational analysis. In WP2 we will transfer splenic CD45.1+ monocytes into fed and fasted mice as described in WP1 for transcriptional analysis. We will analyze CD45.1+ monocytes that were recruited to the bone marrow, as well as CD45.1+ monocytes that remained in the blood circulation or homed to the spleen. The comparison between monocytes that returned to the bone marrow in fed vs. fasted mice will identify specific fasting-induced modifications. We will also include a monocyte sample from the spleen of a fed mouse for analysis of the pre-transfer state. We will enrich CD45.1+ monocytes from the respective organs using the Miltenyi monocyte isolation kit and subsequently use flow cytometry to sort to high purity for sequencing. Because we do not expect to yield high cell numbers, we will use ultra-low input sequencing approaches and multiplex samples to save resources. The computational analysis will include among others: differentially expressed genes between bone marrow and peripheral monocytes during fasting, gene ontology, KEGG and pathway analysis, upstream regulators of transcriptional changes, prediction of modified cellular functions, analysis of cellular metabolic and transcriptional networks as well as intercellular communication using Ingenuity Pathway analysis and R tools as we have done before.1

In summary, we will investigate functional modifications of monocytes in the bone marrow during fasting using a monocyte transfer model in combination with transcriptional analysis. The results could indicate how fasting reduces the pro-inflammatory potential of monocytes and, thereby, prevents inflammatory disease and prolongs healthy life.

Application details

References

  1. Jordan S, Tung N, Casanova-Acebes M, […], Berres ML, Gallagher EJ, Merad M. Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool. Cell. 2019; 178:1102-1114 e1117. doi: 10.1016/j.cell.2019.07.050.
  2. Collins, N, Han SJ, Enamorado M, […], McGavern DB, Schwartzberg PL, Belkaid Y. The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction. Cell. 2019; 178:1088-1101 e1015. doi: 10.1016/j.cell.2019.07.049.
  3. Nagai M, Noguchi R, Takahashi D, […] Takubo K, Dohi T, Hase K. Fasting-Refeeding Impacts Immune Cell Dynamics and Mucosal Immune Responses. Cell. 2019; 178:1072-1087 e1014. doi: 10.1016/j.cell.2019.07.047.

Dissecting tissue-homeostatic functions of group 2 innate lymphoid cells using novel genetic tools

Prinicipal Investigator

Dr. Christoph Klose
Prof. Dr. Andreas Diefenbach

Scientific interest within the context of the graduate college:

Type 2 Inflammation, innate lymphoid cells, tissue-homeostasis, neuro-immune interactions.

Project description:

Type 2 immune responses promote tissue homeostasis as well as tissue remodeling and protect against infections with macroparasites but can become detrimental when triggered against non-infectious environmental stimuli.1 The cytokines IL-25, IL-33, and TSLP are strong activators of type 2 inflammation in tissues via stimulation of group 2 innate lymphoid cells (ILC2s) and other innate immune cells, such as eosinophils, mast cells, basophils, and alternatively activated macrophages resulting in a cytokine milieu, which promotes differentiation of T helper 2 cells and secretion of immunoglobulin E.1,2 Although ILC2s become quickly activated, the precise role in orchestrating type 2 immune responses remains elusive due to the limitations in specifically targeting this population in the presence of adaptive immune cells because of the large overlap in expression of ILC2s with T cells and other immune cells.

To guide over this major limitation in the field, we could recently generate and evaluate a model to specifically deplete ILC2s based on the Nmur1 promoter. Using this model, we could recently show that ILC2s are the major determinate of eosinophil homeostasis in tissues if ILC2 are genetically ablated from the beginning. Exploiting the possibilities of this newly generated tool, we now aim to deplete ILC2 after development (using antibody depletion against ectopically expressed hCD2) or to pharmacologically modulate ILC2 (using ectopically expressed designer drug receptors) functions. The downstream effector functions will also be explored by conditional deletion of interleukin 5 in ILC2. Our working hypothesis proposes that ILC2s could be a promising target via pharmacological modulation or antibody-mediated depletion because of their tissue-homeostatic functions, namely regulation of eosinophils in tissue via IL-5. The models to test this hypothesis are available in our lab, and the techniques are carried out on a daily basis without the requirement to establish novel methods from scratch. Delineating the regulation of type 2 immune responses by ILC2s will be key to understand how type 2 immune responses are orchestrated. Using both focused and global experimental approaches our research has the potential to discover novel molecular pathways, which can be harnessed for the maintenance of homeostasis and prevention of chronic inflammation.

Application details

References

  1. Palm NW, Rosenstein RK, Medzhitov R. Allergic host defences. Nature. 2012; 484:465-472. doi: 10.1038/nature11047.
  2. Klose CSN, Artis D. Innate lymphoid cells control signaling circuits to regulate tissue-specific immunity. Cell Res. 2020; 30:475-491. doi: 10.1038/s41422-020-0323-8.
  3. Moro K, Yamada T, Tanabe M, […], Ohtani M, Fujii H, Koyasu S. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. Nature. 2010; 463:540-544. doi: 10.1038/nature08636.
  4. Nussbaum JC, Van Dyken SJ, von Moltke J, […], Chawla A, Liang HE, Locksley RM. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature. 2013; 502:245-248. doi: 10.1038/nature12526

NF-κB in homeostasis, acute inflammation, and mucosal healing

Prinicipal Investigator

Scientific interest within the context of the graduate college:

Our group “Signal Transduction in Health and Disease” (Department of Gastroenterology and Hepatology, Charité 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. Recent studies showed that crosstalk between epithelium and immune cells changes in health and disease, and that is in part due to altered functions of NF-κB. Our group aims to decipher that crosstalk and identify the changes. Our research is highly interdisciplinary and spans different fields including biochemistry, immunology, stem cell biology, and cancer biology.

Project description:

NF-κB is deemed the master regulator of pro-inflammatory response and its activation directly correlates with severity of inflammation in Inflammatory Bowel Disease (IBD).1 Although in immune cells, NF-κB plays largely pro-inflammatory role, in intestinal epithelium its function is poorly understood. Many IBD therapies rely on suppression of NF-κB. These therapies however inhibit NF-κB across different tissues and cell types, including healthy ones. This can lead to many adverse effects, including susceptibility to infection and carcinogenesis. We have recently shown that a single stress stimulus sequentially activates functionally distinct transcriptomes of NF-κB: anti-apoptotic and pro-inflammatory.2-3 We also showed that in homeostasis, NF-κB directs differentiation of stem cells.4 To identify therapies that specifically target only the pro-inflammatory NF-κB, it is critical to decipher what roles NF-κB plays in health, in acute inflammation, and finally in mucosal healing.

A. Immunofluorescent staining against Sox9, κ-EGFP (activated NF-κB), and DAPI (nucleus) in the small intestine. Only a subset of cells shows activated NF-κB. B. Immunofluorescence of intestinal organoids. Olfactomedin 4 (stem cell marker) and Ki67 (proliferation) are shown. Control depicts littermate controls of IκBα IES KO (intestinal epithelial knockout, which leads to constitutive NF-κB activation). Scale bar: 50 µM.

Based on our recently published and unpublished data we propose that acute, immediate activation of NF-κB in intestinal epithelium is necessary for resolution and for regaining homeostasis. To address this hypothesis we will use single-cell datasets, intestinal organoids, and transgenic mouse models. We have recently established transgenic mice that provide a specific readout of NF-kB activity as well as mice with either specific suppression or constitutive activation of NF-kB in intestinal epithelium.2-4 These tools could enable us, for the first time, to determine where and how NF-κB can be targeted to both prevent chronic inflammation or to promote resolution.

Our key aims are:
1) To identify which cells activate NF-kB under homeostasis versus in inflammation, and to determine which transcriptional programs NF-kB initiates in these cells.
2) To determine how repression or constitutive activation of NF-kB affects intestinal homeostasis, acute inflammation, and resolution.
3) Identify which singling pathways can be targeted to safeguard homeostasis or promote resolution of inflammation.

Application details

References

  1. Atreya I, Atreya R, Neurath MF. NF-kappaB in inflammatory bowel disease. J Intern Med. 2008; 263:591-596. doi: 10.1111/j.1365-2796.2008.01953.x.
  2. Kolesnichenko M, Mikuda N, Höpken UE, […], Schmidt-Ullrich R, Schmitt CA, Scheidereit C. Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence. EMBO J. 2021; 40:e104296. doi: 10.15252/embj.2019104296.
  3. Mikuda N, Schmidt-Ullrich R, Kärgel E, […], Scheidereit C, Kühl AA, Kolesnichenko M. Deficiency in IκBα in the intestinal epithelium leads to spontaneous inflammation and mediates apoptosis in the gut. J Pathol. 2020; 251:160-174. doi: 10.1002/path.5437.
  4. Brischetto C, Krieger K, Klotz C, […], Heuberger J, Scheidereit C, Schmidt-Ullrich R. NF-κB determines Paneth versus goblet cell fate decision in the small intestine. Development. 2021; 148:dev199683. doi: 10.1242/dev.199683.

Deciphering the role of macrophages for allograft quality and survival

Prinicipal Investigator

Prof. Dr. Katja Kotsch
Dr. Arne Sattler
Dr. Efstathios Stamatiades

Scientific interest within the context of the graduate college:

Our group is studying immune-mediated mechanisms of solid organ allograft rejection. Worldwide, the number of available donor organs is decreasing. Moreover, due to demographic changes in modern societies, the number of individuals with an advanced age >65 years is steadily increasing. This results in a higher number of patients diagnosed with progressive chronic kidney disease (CKD) constituting a potential kidney transplant recipient group. Accordingly, chronological donor age is also a major risk factor for allograft dysfunction, as grafts from older donors are more susceptible to ischemic injury and prone to restricted allograft outcome. Despite modern immunosuppressants, overall long-term survival of solid allografts is limited. The infiltration and activation of immune cells is one of the key mechanisms for chronic allograft failure, but the detailed molecular and cellular mechanisms underlying the complex interplay between donor and recipient – finally resulting in the rejection of the graft – are still not understood. Consequently, a better understanding of both, donor organ quality and allograft rejection is mandatory in order to overcome the current limitations of insufficient numbers of donor organs – and restricted allograft survival in the long term.

Project description:

Graft-infiltrating monocyte-derived macroprohages (mdMΦs) post transplantation are phenotypically indistinguishable from kidney-resident macrophages (krMΦs) hampering their discrimination in the graft. In order to explore the important role of krMΦs in graft survival, particularly in terms of donor-related risk factors (e.g. donor age, obesity), we will use state-of-the-art techniques including experimental transplantation models, genetic-fate-mapping, single-cell RNAseq and confocal/intravital/light-sheet microscopy to decipher renal macrophage biology in steady-state and renal transplantation. Experimental animal studies will be complemented by a comprehensive analysis of monocytes/macrophages from patient tissues (resected materials). This project will be embedded in the 2nd funding period of the SFB 1365 (Nephroprotection) at the Charité.

Application details

References

  1. Moreau A, Varey E, Anegon I, Cuturi MC. Effector mechanisms of rejection. Cold Spring Harb Perspect Med. 2013; 3(11):a015461. doi: 10.1101/cshperspect.a015461.
  2. Dornieden T, Sattler A, Pascual Reguant A, […], Boral S, Friedersdorff F, Kotsch K. Signatures and Specificity of Tissue-resident Lymphocytes Identified in Human Renal Peri-tumor and Tumor Tissue. J Am Soc Nephrol. 2021; 32:2223. doi: 10.1681/ASN.2020101528.
  3. Günther J, Resch T, Hackl H, […], Pascher A, Pratschke J, Kotsch K. Identification of the activating cytotoxicity receptor NKG2D as a senescence marker in zero-hour kidney biopsies is indicative for clinical outcome. Kidney Int. 2017; 91:1447-1463. doi: 10.1016/j.kint.2016.12.018.
  4. Stamatiades EG, Tremblay ME, Bohm M, […], Diebold S, Nimmerjahn F, Geissmann F. Immune Monitoring of Trans-endothelial Transport by Kidney-Resident Macrophages. Cell. 2016; 166:991-1003. doi: 10.1016/j.cell.2016.06.058.
  5. Carlin LM*, Stamatiades EG*, Auffray C, […], Cook HT, Diebold S, Geissmann F. Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell. 2013; 153:362-375. doi: 10.1016/j.cell.2013.03.010. *equal contribution.

The role of the primary cilium in the physiological autoregulation of the pulmonary vasculature

Prinicipal Investigator

Prof. Dr. Wolfgang Kübler
Prof. Dr, Robert Szulcek

Scientific interest within the context of the graduate college:

Maintenance or restoration of vascular homeostasis are critical determinants of health throughout life. Of late, the primary cilium has been identified as a key regulator of vascular homeostasis and regeneration. While impairment of primary ciliary structure and signaling can promote vascular disease and vascular remodeling, strategies aiming to preserve or restore ciliary function emerge as novel therapeutic approaches to maintain vascular health.

Project description:

Physiologically, homeostatic signaling in response to biochemical and biomechanical cues within the pulmonary vascular wall warrants the integrity of the vascular structure of the lung and allows for its adaptation to changing requirements throughout life. Dysregulated signaling in or between pulmonary artery endothelial and smooth muscle cells, on the other hand, causes progressive adverse remodeling of the pulmonary vasculature, resulting in increased pulmonary vascular resistance, pulmonary hypertension, and ultimately death due to right heart failure. Understanding the cellular signaling mechanisms that maintain or restore vascular homeostasis in the lung is therefore critical for the prevention or therapy of pulmonary hypertension, and for the preservation of an intact vasculature throughout life up to an old age.

Ongoing research in our group has identified the primary cilium as a novel key regulator of vascular homeostasis in the lung. Unlike the motile cilia found e.g. on the respiratory epithelium or in the Fallopian tube, the primary cilium is a singular organelle (one per each cell) that is found on almost every cell type in the body. The primary cilium extends as a protrusion of the cell membrane into the extracellular space where it functions as a mechano- and chemosensor, while its base acts as a hub for numerous cellular signaling pathways. We have shown that the primary cilium maintains the cells of the pulmonary vasculature in a physiological quiescent state. Loss of the primary cilium, however, causes pathological proliferation and migration of both cell types. Importantly, we have identified such loss of the primary cilium as a hallmark of pulmonary blood vessels in patients with pulmonary hypertension. These findings fuel the intriguing hypothesis that strategies aiming to preserve or restore primary cilium structure and function may present a novel therapeutic approach to maintain lung vascular health and to reverse maladaptive vascular remodeling. In order to test this hypothesis, the present project will address the following questions:

Question 1: Which mechanisms regulate the integrity of the primary cilium in lung vascular endothelial and smooth muscle cells? Based on previous work by us and others, we will focus here specifically on signaling axes via mammalian target of rapamycin (mTOR) and aurora kinase A. Specifically, we will test whether pharmacological or genetic modulation of these pathways can maintain primary ciliary integrity in preclinical models of pulmonary hypertension.

Question 2: Can strategies aimed to restore ciliary integrity preserve lung vascular homeostasis? We have recently patented a novel intervention for rescue of the primary cilium in pulmonary hypertension. We will test whether this strategy, as well as interventions identified in Aim 1 can restore vascular homeostasis in preclinical models of pulmonary hypertension both in vitro and in vivo.

The project will be conducted at the Institute of Physiology at the Campus Mitte under close supervision by the PI and Co-PI and with the support of a dedicated research group incl. several postdocs and technical personnel, as well as several international collaborators in Europe, USA, Canada, and China. All required methods are established. The graduate student is expected to present his/her work at international conferences, and to publish the results as a full manuscript in a major peer-reviewed scientific journal.

Application details

References

  1. Fan Y, Gu X, Zhang J, […], Solymosi P, Kwapiszewska G, Kuebler WM. TWIST1 drives smooth muscle cell proliferation in pulmonary hypertension via loss of GATA-6 and BMPR2. Am J Respir Crit Care Med. 2020; 202: 1283-1296. doi: 10.1164/rccm.201909-1884OC.
  2. Dummer A, Rol N, Szulcek R, […], DeRuiter MC, Goumans MJ, Hierck BP. Endothelial dysfunction in pulmonary arterial hypertension: loss of cilia length regulation upon cytokine stimulation. Pulm Circ. 2018; 8: 2045894018764629.doi: 10.1177/2045894018764629.


Role of mechanical signals for growth and regeneration of bones / LR signaling in aging and joint trauma as a potential cause of osteoarthritis

Prinicipal Investigator

Prof. Dr. Max Löhning
Dr. Maria Dzamukova
Dr. Ping Shen

Scientific interest within the context of the graduate college:

Our laboratory investigates the cellular and molecular causes of osteoarthritis, the most common joint disease in adults worldwide. To date, there is no therapy that alters the course of the disease. The onset of osteoarthritis is closely associated with advanced age and joint trauma, and the signaling pathways involved are still poorly understood. Therefore, we aim to gain a better understanding of the control of cartilage and bone formation and degradation in joints during aging and trauma. We will first study these processes in the healthy state and then compare them with their regulation in osteoarthritis. The focus here is on the cartilage-forming cells, the chondrocytes, as well as the cells of the synovial membrane and the subchondral bone. The changes in cartilage and bone metabolism identified in this way offer potential targets for causal therapies by biological regeneration of articular cartilage.

Project description – Project 1:

Role of mechanical signals for growth and regeneration of bones

In a recent study, we found that mechanical loading regulates bone growth. This is achieved by controlling angiogenesis and bone mineralization in the ossification front – growth plate region and also in subchondral bone via mechanical forces.1 However, how mechanical force is perceived molecularly is still not fully understood. Based on our data and recent publications,2,3 mechano-responsive ion channels of the PIEZO family appear here as promising mechano-receptor candidates. Given the importance of mechanical cues in bone regeneration,4 we aim to investigate the role of PIEZO molecules and their signaling pathways in the regulation of bone growth and bone healing in this project.

Project description – Project 2:

TLR signaling in aging and joint trauma as a potential cause of osteoarthritis

Our current data show that stimulation of specific Toll-like receptors (TLRs) severely impairs the mitochondrial respiratory chain and cartilage production of human chondrocytes (Shen et al., submitted). We hypothesize that age- or joint-trauma-induced degradation processes release cartilage fragments that activate specific TLR molecules. Chondrocytes respond by amplifying their TLR signaling pathways. This causes an energy and metabolic deficit and triggers further cartilage degradation, which could ultimately lead to osteoarthritis. To further investigate changes in TLR signaling and metabolic pathways, we plan to perform RNA sequencing of human chondrocytes from the following donors: 1) young, healthy; 2) young, post-traumatic osteoarthritis; 3) older, healthy; 4) older, age-associated osteoarthritis. Comparisons of the transcriptomes should reveal age-related changes in healthy cartilage as well as shifts in age- and trauma-associated osteoarthritis.

Application details

References

  1. Dzamukova M, Brunner T, Miotla-Zarebska J, […], Schinke T, Vincent T, Löhning M. Mechanical forces couple bone matrix mineralization with inhibition of angiogenesis to limit adolescent bone growth. Nat Commun. 2022; 13:3059. doi: 10.1038/s41467-022-30618-8.
  2. Li X, Han L, Nookaew I, […], Silva MJ, Almeida M, Xiong J. Stimulation of Piezo1 by mechanical signals promotes bone anabolism. eLife. 2019; 8:e49631. doi: 10.7554/eLife.49631.
  3. Sun W, Chi S, Li Y, […], Liu Z, Xiao B, Li Y. The mechanosensitive Piezo1 channel is required for bone formation. eLife. 2019; 8:e47454. doi: 10.7554/eLife.47454.
  4. McDermott AM, Herberg S, Mason DE, […], Kelly DJ, Alsberg E, Boerckel JD. Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration. Sci. Transl. Med. 2019; 11:eaav7756. doi: 10.1126/scitranslmed.aav7756.

The role of Muc5b in lung homeostasis and onset and progression of interstitial lung disease

Prinicipal Investigator

Prof. Dr. Marcus A. Mall
Dr. Julia Dürr

Scientific interest within the context of the graduate college:

Mucociliary clearance is the primary innate defense mechanism of the lung and crucial to maintain lung homeostasis and health. Mucociliary clearance relies on motile cilia on the surface of epithelial cells and a protective mucus gel layer entrapping particles and pathogens to be cleared from the lungs. Recent evidence suggests that the secreted mucin MUC5B that is crucial for the formation of the mucus gel and proper mucociliary clearance is also implicated in the pathogenesis of interstitial lung disease (ILD).1 ILD can affect children and adults and is characterized by interstitial inflammation, rapid progression of pulmonary fibrosis and subsequent disruption of the alveolar gas exchange ultimately leading to respiratory failure. The understanding of the pathogenesis remains limited and ILD is usually diagnosed in advanced stages when irreversible lung damage has already occurred. Further, only limited therapeutic options are available, which so far, cannot prevent progression of pulmonary fibrosis. By conditional deletion of Nedd4-2 (Nedd4-2-/-) in lung epithelial cells of mice, we recently generated the first mouse model that develops spontaneous pulmonary fibrosis sharing key features with ILD patients allowing us to study lung homeostasis at baseline and early dysregulation leading to the development of interstitial lung disease.2,3

Project description:

The aim of this translational research project is to investigate the role of Muc5b in the onset and progression of interstitial lung disease in conditional Nedd4-2-/- mice. For this purpose, double knockout mice deficient for Nedd4‑2 and Muc5b will be generated and their lung phenotype comprehensively studied at different stages of disease progression to identify early changes in lung homeostasis and pathways that promote disease progression and may serve as therapeutic targets. Clinical parameters such as inflammation markers, histology, lung function, as well as mucus properties will be investigated in order to gain insight into the relationship between altered mucus properties and disease pathogenesis. Further, our findings will be validated in lung biopsies and serum samples from patients with ILD. In addition to basic molecular biology laboratory work, this experimental MD thesis also applies state-of-the-art mouse lung function, mucus rheological measurements, and high content microscopy.

Application details

References

  1. Evans CM, Fingerlin TE, Schwarz MI, […], Warg L, Yang IV, Schwartz DA. Idiopathic Pulmonary Fibrosis: A Genetic Disease That Involves Mucociliary Dysfunction of the Peripheral Airways. Physiol Rev. 2016; 96:1567-1591. doi: 10.1152/physrev.00004.2016.
  2. Duerr J, Leitz DHW, Szczygiel M, […], Beers MF, Klingmüller U, Mall MA. Conditional deletion of Nedd4-2 in lung epithelial cells causes progressive pulmonary fibrosis in adult mice. Nat Commun. 2020; 11:2012. doi: 10.1038/s41467-020-15743-6.
  3. Leitz DHW., Duerr J, Mulugeta S, […], Dalpke AH, Beers MF, Mall MA. Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice. Int J Mol Sci. 2021; 22:6146. doi: 10.3390/ijms22116146.

Neuro-immune interactions in the GI tract: Control of gut homeostasis and function by intestinal regulatory T (Treg) cell-derived endogenous opiods

Prinicipal 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. Because mucosal surfaces are constantly challenged by fluctuating environmental perturbations, immune cells at these sites display a remarkable adaptive capacity in order to fend off microbial challenges and safeguard organ homeostasis and health.

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

Project description:

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, can activate opioid receptors on the enteric circuitry to control crucial physiological functions such as gut motility and secretion. The importance of opioids in regulating gut homeostasis is illustrated by the adverse effects associated with pharmacological opioid intervention during pain therapy, such as Opioid-induced bowel dysfunction (OIBD) that is commonly described as constipation. Vice versa, therapeutic administration of exogenous opioids is widely used to manage severe diarrhea as well as irritable bowel syndrome. Thus, the amount of bioavailable opioid peptides in the GI tract is strictly determining gut function and health.

Throughout the last years, gut-resident Foxp3+ Treg cells have been associated with a growing number of tissue-specific functions in the intestine, comprising various aspects of gut immunity and physiology. Treg cells have pivotal roles in intestinal tolerance induction and host defense by actively controlling immune responses towards dietary antigens and commensal microorganisms as well as towards invading pathogens. In addition to these cardinal immune-related roles, it has become increasingly clear that 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 a specific cellular source of endogenous opioid peptides in the gut. Since intestinal Treg cells are known to incorporate diverse external signals in the GI tract, such as signals derived from the diet or microbiota, their potential involvement in the opioid-mediated control of gut function may represent a key mechanism of tissue adaptation to constantly changing environmental cues.

Aim 1: Phenotypic, functional and spatial characterization of opioid-releasing Treg cells in the intestine. Intestinal Foxp3+ Treg cells are a heterogenous cell population, comprising of distinct subsets with different developmental origins, functions and phenotypes, tailored to the diverse challenges of the intestinal tissue microenvironment. Therefore, in Aim 1, we plan to thoroughly characterize murine opioid-releasing 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-releasing 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-releasing Treg cells and their potential interaction partners in the murine gut.

Aim 2: Identification of signals regulating opioid-releasing 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. cytokines, dietary signals, and microbiota) 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-releasing Treg cells for gut physiology and homeostasis. To ultimately test the role of Treg cell-derived opioids for gut homeostasis and function, we have generated conditional T cell and Treg cell-specific opioid knockout mice. Hence, in Aim 3, we will use these mice to perform functional analysis 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.

Application details

References

  1. Cosovanu C, Neumann C. The Many Functions of Foxp3+ Regulatory T Cells in the Intestine. Front Immunol. 2020; 11:600973. doi: 10.3389/fimmu.2020.600973.

Epigenetic mechanisms in T cell senescence and aging

Prinicipal Investigator

Scientific interest within the context of the graduate college:

The three-dimensional structure of the genome – the epigenome – contributes significantly to the regulation of gene expression and forms the molecular basis for each cell type-specific transcription profile. In contrast to the DNA sequence, however, the epigenome can be modified by external influences and can therefore adapt the gene expression profile of a cell to new circumstances (e.g. in the case of differentiation, aging, environmental influences).

Our laboratory investigates the epigenetic regulatory mechanisms in immune cells, which have to react particularly flexibly to external influences (infection, inflammation, microbiota, aging, etc.). We profile genome-wide epigenetic structures to identify key elements that make a decisive contribution to the generation and function of immune cells during health1 and in situations of misguided immune reactions (e.g. chronic inflammation and autoimmunity). These elements will help to clarify the molecular reasons for misregulated immune reactions and might represent promising therapeutic targets. Furthermore, in situations where immune cells serve as therapeutic agents to regain health (advanced therapy medicinal products, ATMPs, such as adoptive cell therapy) epigenetic structures may be used as biomarkers for quality and safety control purposes2, and also as molecular switches for gene expression (‘epigenetic editing1,3), which may equip cell products with desired characteristics.

Project description:

The immune system, and especially T lymphocytes, go through aging processes that contribute to a reduced effectiveness of the immune system in old age (so-called: immune aging). Epigenetic mechanisms also contribute to immune aging. We were able to show that characteristic changes occur in the DNA methylation profile of T cells, which appear to be caused by proliferation-induced senescence processes.1,4 Interestingly, a large part of these changes occur in the transcriptionally silent part of the genome, the heterochromatin, in which genes are packaged for permanent repression. In this project, we want to generate genome-wide DNA methylation profiles of various T cell subsets specific for acute or chronic viruses in young and elderly people. Based on these molecular signatures, we want to clarify the DNA methylation changes, which are induced by proliferation-induced senescence during acute or chronic antigen exposure as well as during physiological aging of the human body. These results might also be of relevance for autoimmune and chronic inflammatory diseases as the activation and proliferation history of T cells accumulates in patients over time due to chronic or repetitive antigen encounter. Furthermore, this study will also reveal potential molecular targets for the rejuvenation of T cells, which might be useful for the application of T lymphocytes as ATMPs. This project requires the application of various methods from cell biology (e.g. flow cytometry, T cell isolation and culture), molecular biology (e.g. DNA methylation profiling) as well as bioinformatics (e.g. analysis of array-based genome-wide DNA methylation profiles), all of which are established in our lab and can be learned by the doctoral student.

Application details

References

  1. Durek P, Nordström K, Gasparoni G, […], Walter J, Hamann A, Polansky JK. Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development. Immunity. 2016; 45:1148-1161, doi: 10.1016/j.immuni.2016.10.022.
  2. Ou, K, Hamo D, Schulze A, […], Schmueck-Henneresse M, Reinke P, Polansky, JK.Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function. Front Cell Dev Biol. 2021; 9:751590. doi: 10.3389/fcell.2021.751590.
  3. Kressler C, Gasparoni G, Nordström K, […], Walter J, Hamann A, Polansky JK. Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype. Front Immunol. 2020; 11:609891. doi: 10.3389/fimmu.2020.609891.
  4. Salhab A, Nordström K, Gasparoni G, […], Lengauer T, Manke T, DEEP Consortium, Walter J. A comprehensive analysis of 195 DNA methylomes reveals shared and cell-specific features of partially methylated domains. Genome Biol. 2018; 19:150, doi: 10.1186/s13059-018-1510-5.