The gut-liver-axis - functional circuits and therapeutic targets in health and disease
Gut and liver act as a functional entity in health and disease. They develop from a common cellular ancestor during ontogeny, cooperatively facilitate food uptake and metabolism, structurally and functionally separate the microbiota-containing gut lumen from the systemic circulation, and act in concert to ensure immunity to food-born pathogens. The major aim of this IZKF Joint Research Project is to replace the traditional organ centered view by an integrated approach considering the combined function of gut, liver, their metabolic capacity and the microbiota.
A number of anatomical, cellular and molecular circuits facilitate the cooperative action of both organs. The portal vein channels venous blood drained from the intestine to the liver and conversely the bile duct transports hepatic products and mediators into the intestinal lumen. Endocrine mediators, immune cells migrating along similar trails and microbiota-derived immune stimuli functionally interconnect both organs. The IZKF Joint Research Project therefore includes a multidisciplinary group of researchers to address the many aspects of this cooperative organ function
The functional link between gut and liver also becomes apparent in clinical medicine reflected by a number of combined manifestations in settings of intestinal and hepatic dysfunction and disease The clinical implications of this coordinated action, however, have not been thoroughly investigated but could provide novel and innovative targets in future patient care. We therefore combine clinical oriented studies with basic research in order to find new diagnostic but also interventional strategies for the future treatment of affected patients.
Research Area Inflammation and Consequences - Projects
Here you find the summarys of the subprojects of the Gut Liver Joint Research Initiative.
P01 Microbiota and TLR-signalling in the Development and Recurrence of intrahepatic Cholangiocarcinoma
PI: Univ.-Prof. Dr. Tom Lüdde, Division of Gastroenterology, Hepatology and Hepatobiliary Oncology
The gut microbiome plays a fundamental role in the modulation of the hepatic microenvironment, a function that is essential in the progression from chronic hepatic inflammation to liver fibrosis/cirrhosis and eventually hepatocellular carcinoma (HCC), the most frequent primary liver cancer.
Recently, microbial components and a dysbiosis of the intestinal and biliary microflora were proposed as potential triggers and modulators of benign biliary diseases, but the exact roles of microbiota and toll-like-receptor (TLR)-signalling in intrahepatic cholangiocarcinoma (iCCA) – a malignant intrahepatic tumour of biliary differentiation with rising incidence and limited treatment options – have remained elusive.
In this present proposal, we therefore plan to:
- Evaluate if and how signals triggered by the intestinal microbiome via distinct TLR-dependent signalling cascades modulate the development of iCCA in a genetic murine tumour model.
- Examine the role of specific components of the gut microbiota in the recurrence of iCCA after surgical resection as a proof-of-principle for new adjuvant treatment strategies.
- Transfer the findings from murine disease models into human patients using a recently established serum data bank and patient specimens from an interventional clinical trial (ARROW-Trial) on selective microbiome-modification in liver tumour patients.
We aim to establish a new functional concept how microbiome-associated signalling pathways modulate iCCA development and recurrence and providing a proof-of-concept for microbiome-modulation in the adjuvant treatment of resected iCCA.
P02 Tissue regeneration as a critical component of gut-liver homeostasis
PI: Dr. nat.med. Yulia A. Nevzorova , Department of Internal Medicine III
Co PI: Prof. Dr. rer.nat. Christian Liedtke , Department of Internal Medicine III
The intestinal epithelium is characterized by a high cell turnover involving proliferation of progenitor cells and apoptosis of differentiated enterocytes. Any disturbance of this homeostasis e.g. through impaired proliferation or by toxic agents may result in increased intestinal permeability, translocation of bacterial products from the gut to the liver and secondarily to chronic liver disease.
E-type Cyclins (Cyclin E1, Cyclin E2) are regulatory subunits of the Cyclin-dependent kinase 2 (Cdk2) and mediate the transition of resting cells into cell cycle progression. In the last decade we have uncovered several important functions of Cyclin E1 in the setting of chronic hepatic and intestinal injury.
We provided evidence that Cyclin E1 is essential for initiation of liver fibrosis and hepatocellular carcinoma and plays a fundamental role for the prevention of intestinal permeability in the setting of excessive alcohol challenge. In this project we hypothesize that the Cyclin E/Cdk2 complex is important for the maintenance of a functional intestinal barrier and aim to determine the consequences of a de-regulated cell cycle for the intestinal permeability and subsequent crosstalk with the liver.
The following aims will be addressed:
- To dissect the functions and contributions of E-type Cyclins and Cdk2 for basal prevention of gut permeability and inflammatory gut-liver crosstalks.
- To analyse the role of alcohol for cell cycle regulation and CcnE1-interactome formation in the intestine, which can have a direct implication for communication with the liver.
- To define the contributions of E-type cyclins for initiation of colorectal cancer and progression of derived liver metastases.
In summary this project will identify the essential core cell cycle machinery in the intestine required for barrier homeostasis and balanced gut-liver interactions.
P03 Identification of mediators involved in the gut-liver-cross-talk in portal vein blood from patients with portal hypertension using a plasma proteomic approach
PI: Heidi Noels, PhD, Institute for Molecular Cardiovascular Research
Co-PI: Univ.-Prof. Dr. med. Christiane Kuhl, Department of Diagnostic and Interventional Radiology
Portal hypertension is one of the most serious sequelae of chronic liver disease. Different therapeutic options such as “transjugular intrahepatic portosystemic shunts” (TIPS) treatment have been developed; however, complications of portal hypertension still cause significant morbidity and mortality. Being the main supplier of hepatic blood flow, the portal vein serves as a “superhighway” for gut-derived mediators to the liver. The knowledge of these mediators is essential for our understanding of the gut-liver cross-talk in both the physiological and pathophysiological situation, aiming at a targeted therapy for liver disease.
Furthermore, portal hypertension is diagnosed during hepatic vein catheterisation as well endoscopy as a gold standard, but the latter is not available in every clinic and holds a high cost and many potential risks. This highlights the strong need for plasma mediators and biomarkers to be translated into an innovative, minimally invasive diagnostic tool to identify patients with portal hypertension.
Supported by our preparatory data and using blood samples from patients with portal hypertension undergoing TIPS treatment and a combination of plasma proteomics and functional bio-assays, we aim to
- identify and characterise gut-derived molecular determinants associated with portal hypertension and adversely affecting liver pathology, as they represent interesting therapeutic targets in portal hypertension-associated liver disease; and
- identify distinct molecular determinants in systemic blood of patients with portal hypertension, as they represent potential biomarkers of portal hypertension using a minimally-invasive diagnostic method.
Ultimately, this project may result in an improvement of both therapy and diagnostics of portal hypertension, thereby reducing the burden of patients.
P04 Disturbed enterohepatic bile salt signaling and consequences for liver regeneration.
PI: Dr. Frank G. Schaap, Uniklinik RWTH Aachen, Department of General, Visceral and Transplantation Surgery
Co-PI: Prof. Dr. Steven W.M. Olde Damink, Uniklinik RWTH Aachen, Department of General, Visceral and Transplantation Surgery
Liver resection exploits the regenerative potential of the liver and represents in principal a safe and curative procedure. Post-resectional liver failure is the consequence of insufficient remnant liver function to sustain vital liver functions during the regenerative phase. Perturbation of the enterohepatic circulation is thought to impair liver regeneration in patients undergoing liver resection, by derangement of bile salt signaling. In this project we will test the hypothesis that disruption of bile salt-induced mitogenic activity, combined with dysregulated bile salt homeostasis and associated hepatotoxicity, results in impaired liver regeneration and consequently postoperative morbidity and mortality in (post-) cholestatic patients undergoing liver resection. By extension, we will analyze whether post-resectional liver failure may be counteracted by agonistic activation of the bile salt receptor FXR, or therapeutic administration of FGF19, an FXR regulated enterokine with bile salt-homeostatic and mitogenic activity. Specific objectives are:
- To determine enterohepatic fluxes and organ sources of bile salts and regeneration-related signaling molecules in patients with normal and abrogated enterohepatic circulation, under basal and prandial conditions.
- To characterize bile salt signaling in enterohepatic tissues and liver cell populations of patients with normal and abrogated enterohepatic circulation.
- To assess liver regeneration in an ex vivo model employing tissue from non-cholestatic andinitially cholestatic patients receiving pre-operative biliary drainage (post-cholestatic state).
- To determine the effect of the antibiotic Rifaximin on bile salt signaling in patients undergoing major liver resection.
- To test therapeutic strategies in animal models of post-resectional liver failure.
The combined animal and patient studies are expected to provide functional insight in the role of bile salt signaling and bile salt toxicity, as well as the contribution of the gut microbiome and innate immunity during normal and defective liver regeneration after partial hepatectomy, enabling novel therapeutic approaches.
P05 Impact of bile acid conversion by Coriobacteriaceae on the gut-liver axis
PI: Prof. Thomas Clavel, PhD, Institute of Medical Microbiology
In spite of numerous observations that shifts in gut bacterial communities are linked to a variety of chronic diseases, including liver pathologies such as steatosis or non-alcoholic steatohepatitis, there is a lack of targeted studies providing insight into underlying molecular mechanisms. Bile acids produced in the liver and released in the small intestine to facilitate lipid absorption represent paradigm metabolites illustrating the important influence of microbial functions on host physiology. Indeed, specific gut bacteria can deconjugate, dehydroxylate, or dehydrogenate bile acids, and thus markedly alter their bioavailability and bioactivities (e.g. modified hydrophobicity, toxicity, or receptor affinity). One family of bile acid-converting bacteria is the Coriobacteriaceae (CORIO), which includes dominant and prevalent members of mammalian gut microbiota. Although these bacteria are normal dwellers of the human intestine in healthy subjects, their occurrence has also been associated with a variety of diseases. Literature and own data suggest that CORIO modulate host lipid metabolism and that their occurrence is associated with hypercholesterolemia. However, the underlying mechanisms are not defined and the impact on liver physiology, as central organ for lipid and bile acid homeostasis, is unknown. Hence, we will perform targeted mechanistic studies in gnotobiotic models to address the following issues:
- Establish a new gnotobiotic model for the mechanistic study of bacterial bile acid conversion by gut bacteria
- Assess diet- and bile acid-dependent responses of the host and the gut bacterial communities.
- Determine the gut and liver responses to CORIO.
Altogether, the project will deliver novel insights into to the causal role of specific gut bacteria in regulation of the gut-liver axis.
P06 Functional role of gut microbiota on PNPLA3-dependent liver disease progression
PI: Prof. Dr. med. Christian Trautwein, Medizinische Klinik III, Uniklinik, RWTH Aachen
Co-PI: Dr. med. Maximilian Hatting, Medizinische Klinik III, Uniklinik, RWTH Aachen
Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a triacylglycerol lipase in several lipid metabolizing tissues such as hepatocytes. A polymorphism of PNPLA3 at position 148 (I148M) as regularly found in the Caucasian population is a risk factor for metabolic liver disease and other liver conditions, suggesting that PNPLA3 has a function in liver homeostasis that goes beyond lipid storage modulation.
In this project, we will study the impact of PNPLA3 I148M on gut liver communication and consequences for liver disease onset and progression. Defining these changes in a translational approach will significantly contribute to a better understanding of liver disease progression and thus identify novel diagnostic and therapeutic targets.
P07 Enterohepatic cooperation in the postnatal establishment of immune homeostasis
PI: Prof. Dr. med Mathias Hornef, Institute of Medical Microbiology
The postnatal period represents a critical time window for the establishment of the enteric microbiota and a stable intestinal host-microbial homeostasis. Concomitantly, the liver undergoes functional maturation from a primary hematopoietic organ to a central organ of metabolic regulation and immune surveillance. Together, the intestinal mucosa and the liver shape the intestinal luminal environment and form the barrier to shield the systemic circulation from gut-derived microbial and dietary stimuli. However, the structural and functional mechanisms that generate this barrier and shape the enteric microbiota in the neonatal intestine to facilitate a stable host-microbial homeostasis have not been investigated. The present study aims at the identification and characterization of both adaptive and developmental mechanisms that promote and shape bacterial colonization and prevent an inappropriate systemic immune response to postnatal microbial exposure. We will address the following specific aims:
- Define the transcriptional response of the hepatic tissue to postnatal microbial exposure and identify regulatory mechanisms that maintain hepatic and mucosal immune homeostasis.
- Determine the contribution of excretory functions of the liver to intestinal barrier integrity and the establishment of host-microbial homeostasis.
- Characterize the effect of the neonatal gut-liver barrier on the systemic immune response.
The identification and characterization of mechanisms that establish the postnatal immune homeostasis and coordinate the enterohepatic barrier function is will provide new insights in the critical role of the immediate postnatal period for organ function and disease susceptibility.
P08 Contribution of gut tropic cell adhesion molecules to gut/liver communication during homeostasis and inflammation
PI: Prof. Dr. med. Norbert Wagner, Dept. of Pediatrics, University Hospital RWTH Aachen
Co-PI: Dr. med. Daniela Kroy, Dept. of Internal Medicine III, University Hospital RWTH Aachen
Directed migration of immune cells is a prerequisite for the regulation of immune responses and is secured by the specificity and interactions of chemokines and adhesion molecules (AM). As a therapeutic approach, selective inhibition of immune cell migration has already reached the stage of clinical application. We have shown that the AM β7 integrin, L-selectin, and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) contribute to development of gut-associated lymphoid tissue (GALT), intestinal lymphocyte homing, oral tolerance, inflammatory bowel disease (IBD), and non-alcoholic steatohepatitis (NASH). As immune cell migration into gut and liver shares some of the AM involved and hepatic diseases can develop as extra-intestinal manifestations of IBD, an immunological relation between the intestine and the liver is suggested. We want to delineate the contribution of gut-tropic AM to homeostasis and inflammation of the liver and to the interdependence of intestine and liver immune responses during intestinal or hepatic inflammation. In particular, the aims of this project will be:
- To define the role of gut-tropic AM in hepatitis, fatty liver disease, and alcoholic liver disease (ALD) in mice and in humans and to identify disease promoting AM dependent immune cell types
- To define AM dependent crosstalk of liver and gut during hepatitis and IBD
- To define critical intracellular interaction domains for integrin function with respect to liver and gut disease
P09 Dendritic cells as cellular target of the gut – liver communication in chronic liver disease and hepatocellular carcinogenesis
PI: Dr. med. Marie-Luise Berres, Department of Internal Medicine
Dendritic cells are highly potent antigen-presenting cells governing innate immune responses in the steady state, inflammation, infections, as well as chronic diseases and carcinogenesis. In the liver, DC are constantly exposed to factors derived from portal venous blood draining the intestine and might even be recruited from circulating cells within the portal vein. Due to various factors such as portal hypertension, patients with chronic liver disease (CLD) exert an increased intestinal permeability associated with local inflammation and translocation of microbial products to the liver via the portal vein able to modulate the intrahepatic microenvironment.
In this project we will:
- Comprehensively characterize the intrahepatic DC immunophenotype in patients with CLD with or without HCC as well as in experimental mouse models of HCC
- Assess the impact of intestinal passage of DC progenitors, gut microbiota and intestinal permeability on intrahepatic DC subsets, maturation status, and compartmental distribution in human and mice
- Characterize in vivo the functional impact of increased intestinal permeability and microflora alteration on CLD and HCC progression mediated by dendritic cells
These studies will provide us with a detailed understanding how alteration of the gut microenvironment and increased intestinal permeability during chronic liver disease impact differentiation and maturation of intrahepatic DC and consecutively modulate anti-tumorigenic immune responses. The results might set the stage to generate novel therapeutic regimes combining immunomodulatory with tumor cell directed strategies to improve efficacy of the current therapies and overall outcome of patients with CLD and HCC.
P10 Liver Dendritic Cells in the Induction of Oral Tolerance
PI: Dr. Vuk Cerovic, Ph.D. , Institute of Molecular Medicine, University Hospital RWTH Aachen
Oral tolerance is a crucial process by which the immune system maintains unresponsiveness to harmless intestinal antigens, and prevents unwanted inflammation, such as food allergy or inflammatory bowel disease. The immune systems of the gut and liver cooperate in the induction of oral tolerance by inducing the differentiation of regulatory T cells (Tregs), which then migrate to peripheral tissues where they inhibit inflammatory immune responses. However, while intestinal dendritic cells (DCs) have a well-recognised role in tolerogenic responses, the role of liver DCs has not been characterized in detail, in part due to difficulties in isolation. In this project we aim:
- To characterise the liver DCs for their phenotypic, migratory and functional properties with regards to induction of tolerogenic responses in the liver and gut
- To define the functional and migratory properties of Foxp3+ Treg cells induced by intestinal and liver DCs in the context of oral tolerance
- To examine how the tolerogenic function of DCs and the generation of peripheral Foxp3+ Tregs are altered in conditions of disrupted intestinal homeostasis.
In order to provide a detailed phenotypic and functional characterisation of hepatic DCs, we have established a novel surgical technique to isolate DCs directly from mouse hepatic lymph. In particular, we will assess their capacity for induction of regulatory T cells both in vitro as well as in vivo, following intralymphatic injection.
Furthermore, we will utilise novel transgenic mice expressing a photoconvertible tag to assess, in vivo, the trafficking of Tregs elicited by hepatic and intestinal DCs. Finally, we will examine how the tolerogenic properties of hepatic DCs change under conditions of disrupted intestinal homeostasis, such as increased intestinal permeability, dysbiosis or inflammation.
These approaches will clarify the role of hepatic DCs in the induction of tolerance to intestinal antigens and provide new insights on how the gut and liver cooperate to control tolerance.
P11 Impact of gut-liver communication on hepatic macrophage composition and function
PI: Univ.-Prof. Dr. Frank Tacke, PhD, MHBA, Dept. of Medicine III
Liver macrophages comprise ontogenetically and functionally different subsets. Tissue-resident Kupffer cells and infiltrating monocyte-derived macrophages are key players in the maintenance of immune homeostasis and regulation of inflammatory responses, respectively. We hypothesize that alterations of the gut-liver axis (e.g., altered gut microbiome, intestinal barrier integrity and portal endotoxinemia) affect the composition and function of hepatic macrophages. This is supported by experimental evidence that the gut-liver axis controls macrophage activation in the liver (e.g., using antibiotic treatment or TLR4-ko mice in classic liver disease models), but the mechanisms of hepatic macrophage subset modulation by gut-derived signals is currently obscure. In this project we will:
- Define the impact of gut-derived signals on the origin and differentiation of hepatic macrophage subsets in homeostasis and inflammation.
- Identify the functional consequences of the gut-liver axis on macrophage-driven immune mechanisms in homeostasis and liver diseases.
- Characterize the impact of bacterial translocation on monocyte activation in patients with or without liver diseases.
For these goals, the gut-liver axis will be systematically modified (e.g., rifaximin, mono-colonization, DSS colitis) in healthy mice and mice undergoing liver injury models. We will use innovative methodology including intravital microscopy of liver, single-cell RNA sequencing of hepatic macrophage subsets and a novel 3D-microchip liver model.
The IZKF funding is needed to generate data on the principal mechanisms of the gut-liver axis on hepatic macrophage composition and function and to strengthen collaborations in preparation for the planned SFB.
P12 Secretory antibodies in gut-liver communication
PI: Prof. Dr. rer. nat. Oliver Pabst, Institute of Molecular Medicine
Secretory antibodies are a hallmark of the gut immune system and shield the epithelium from luminal antigens including the gut microbiota. These antibodies derive from different sources: Local production in the intestinal lamina propria and transepithelial transport in the gut, hepatic secretion of Ig-containing bile, and in early life maternal antibodies. In this project, we will integrate the function of antibodies into the coordination of the gut-liver axis and the containment of intestinal antigens.
- describe the clonal relationship of hepatic and intestinal plasma cells,
- define their origin by tracking plasma blast migration from inductive compartments into gut and liver, and
- discriminate the function of intestinal and biliary Ig for the control of intestinal antigen and microbiota.
We expect that our findings will cast a comprehensive picture of how secretory antibodies contribute to the coordination of the gut-liver axis in health and disease.
P13 Noninvasive multimodal imaging of the gut barrier and gut-liver communication
PI: Dr. rer. medic. Dipl.-Inf. Felix Gremse , Institute for Experimental Molecular Imaging
Co-PI: Dr. rer. nat. Wiltrud Lederle, Institute for Experimental Molecular Imaging
Fluorescence-mediated tomography (FMT) enables sensitive and longitudinal assessment of the whole-body distribution of fluorescently labeled probes in mice. The anatomical data provided by µCT (micro-computed tomography) allows improved fluorescence reconstruction, organ segmentation, and generation of organ accumulation curves. In this project, we aim to establish the combination of FMT with MRI (magnetic resonance imaging), which provides higher soft tissue contrast compared to µCT, for imaging the gut-liver axis.
- This technology will be used to assess the permeability of the gut vasculature in healthy and pathologic states (DSS-colitis) with respect to molecules of different sizes.
- To assess gut leakiness, fluorescent probes will be administered via oral gavage and accumulation measured in regions outside the gut.
- To assess bile-related transport between liver and gut, ICG and Gd-EOB-DTPA will be applied intravenously and imaged using dynamic FMT and MRI, since both contrast agents undergo rapid hepatobiliary elimination. This will be performed in healthy mice and mice under alcohol- and high-fat-diet-induced liver diseases.
- Accurate and automated organ segmentation will be implemented using multi-parametric (T1, T2, DWI) MRI sequences to enable efficient and user-independent analysis.
Furthermore, kinetic models will be developed for all experiments to derive robust and physiological parameters from the organ curves. The technology will be established using the mentioned mouse models and used for characterization of novel mouse models in cooperation with the other partners of the consortium.
P14 Investigating the contribution of the intestine microbiome in bile acid metabolism
PI: Prof. Dr. med Ulf Neumann General, Visceral and Transplant Surgery
Co-PI: Prof. Dr. rer. nat. Lars Blank, Institute of Applied Microbiology
The role of the intestinal microbiome and its influence on the host has gained more and more attention in recent years. Intestinal deconjugation of primary bile acids is a key step in enterohepatic circulation of bile acids and largely determines composition of the bile acid pool and consequently its partitioning between plasma and tissue in the body. Recently, we have built a physiologically-based computational model of human bile acid metabolism which describes synthesis, circulation and excretion of representative primary bile acids. The computational model is largely based on physiologically-based pharmacokinetic (PBPK) modelling to account for body anthropometry and physiology. The existing model of primary bile acid metabolism was carefully validated with literature data and is amongst others able to reproduce various key physiological parameters such as transition times of bile acids in the body and in the intestine, respectively, as well as plasma and tissue concentrations of primary bile acids.
In the proposed project, the existing computational model of human will be sophisticated and used in combination with in vitro and in vivo experimental data of the consortium partners in order to better understand and quantify the contribution of the intestinal microbiome in bile acid metabolism.
In particular we have the following three aims:
- consideration of secondary bile acids. This extension of the existing computational model will in particular involve deconjugation reactions in the intestine.
- extrapolation to mice. The human bile acid metabolism model will subsequently be translated to mice to allow the integration of data from the mice experiments of the project partners.
- integration of the gut microbiome. A stoichiometric model of intestinal microbiome will bedeveloped representing the biochemical reactions of the intestinal microbiome at genome scale. The metabolic network model will be linked to the whole-body bile acid model describing enterohepatic circulation.
The final computational model will simultaneously include a description of the metabolic network in the intestinal microbiome as well as a representation of enterohepatic circulation of primary and secondary bile acids at the whole-body level. The model will ultimately be used to characterize diseased states in bile acid metabolism including pathophysiological alterations in the host as well as eubiotic and dysbiotic states of human intestinal microbiome. Furthermore it will provide a platform for the contextualization and analysis of experimental data from the project partners to generate testable hypotheses. The computational model will support a mechanistic understanding of the contribution of gut microbiome in bile acid metabolism to enable the development novel concepts for intervention therapies in the future.
P15 Computational detection of cell sub-populations in the gut and liver from single cell RNA sequencing
PI: Dr. rer. nat. Ivan G. Costa, IZKF Research Group Bioinformatics & Institute for Computational Genomics
The combination of single cell isolation with sequencing techniques permits the study of transcription in a single cell level. This has allowed the characterization of complex organs like liver and gut in homeostatic conditions. The increase in cell throughput and decrease in cost per cell permit the comparison of single cell expression from multiple biological conditions. However, there are so far no computational methods tailored for detection and comparison of population changes of rare cells. Rare cells are of particular importance in the study of gut and liver, where hepatocytes and enterocytes dominate the cell populations. Yet, biologically relevant changes are likely to occur in rare cells such as immune, progenitor and secretory cells. Moreover, existing computational methods do not address technical issues arising from scRNA-seq protocols with high cell throughput, like the high amount of missing data and failure in capturing single cells (multiplets).
This project has three objectives:
(i.) the development of computational methods for finding sub-groups of cells and comparing changes in populations between two or more biological conditions;
(ii.) design of computational strategies for coping with missing data and multiplets events; and
(iii.) analyse population and transcriptional changes during gut ontogeny and liver/gut inflammation.
These will allow us to dissect populations and transcriptional changes of rare cells, such as immune cells and gut secretory cells, during inflammation and infections.