i2TRON - PhD Training Program

Individual projects - Work package 1: Chronic Inflammation

Project 1.1. - Obesity heritage: Generation spanning effect on immunometabolism and inflammation

Introduction

Obesity is one of the biggest societal and occupational health problems worldwide. About one third of the world’s population are considered to be obese. It is well established that obesity fosters the development of several co-morbidities including inflammatory autoimmune disease and cancer, which are tightly linked to a dysregulated immune system. However, whether obesity mediated-changes can be transferred from the obese parents to the next generation and whether this impacts the following generation’s immune system is currently unknown.

Aim and Hypothesis

We hypothesize that parental obesity during pregnancy alters the functionality of the immune system and the susceptibility to inflammatory diseases of the new born child. Especially, we expect changes in the metabolic setup of immune cells, which we aim to characterize within this proposal.

Methods

We will use high fat diet mouse models and in combination in vivo disease models of inflammatory diseases for neuroinflammation, infections and cancer. We aim for a detailed study of the metabolism of immune cells by methods like: metabolic profiling, targeted metabolic tracing, seahorse analysis, RNAseq, etc. Our findings from mouse studies will be validated in human immune cells derived from individuals that had obese parents at the time of their birth.

Project 1.2. - Markers of shaping and educating the early life immune response to food proteins

Introduction

Food allergy (FA) has emerged over the past decade as an important public health concern, culminating in a second wave of the 'allergy epidemic' and thereby succeeding the first respiratory epidemic. Today, more than 17 million Europeans are affected by FA, defined as chronic immune-mediated adverse reactions to otherwise harmless food proteins. The clinical management relies on a strict allergen avoidance. Early-life events, environmental and lifestyle factors play a crucial role in the development of food allergies. The understanding of how those factors synergize in the breakdown of oral tolerance is vital for the foundation of future strategies of food allergy prevention.

Aim and Hypothesis

To address the mechanism of early-life events that are fundamental for shaping the immunological response to food antigens, studying food allergy and tolerance as a disease models. During first years of life, deep immune and genomic profiles as well as microbiome signatures reveal response patterns driving allergic sensitization and clinical food allergy.

Methods

Pregnant women will be included at the participating hospital. Blood will be sampled longitudinally (prebirth to 2 years of age) for detailed analyses of the immune response, the functional microbiome and genetic factors related to food allergy pathogenesis. Integrative modelling of time-resolved clinical metadata, immune profiling data and microbiome multi-omic data will be performed to identify drivers of clinical food allergy. Together with genome datasets, this will allow building personalized, condition-specific or generalizable models to make child-specific predictions about clinical phenotype and potential clinical outcome.

Project 1.3. - Dietary fiber, markers of inflammation and gut microbiota

Introduction

Low dietary fiber intake is a dietary and health concern. Insufficient intake (<25 and 36 g/d, f/m) is frequent in many developed countries including Luxembourg (Alkerwi et al., 2012), and has been related to increased risk of developing various cardio-metabolic complications and higher mortality (McRae et al., 2017) in addition to cancer (Kunzmann et al., 2015). Fiber intake lowers blood lipids (including cholesterol) and is related to anti-inflammatory reactions, fostering a more healthy gut microbiota, with positive systemic effects. However, there is little information to which extent the observed effects are depending on the genetic background of the host (Esworthy et al., 2010). Whole genome sequencing will allow a detailed in-depth investigation of SNPs and other mutations related to the effects of fiber intake.

Aim and Hypothesis

It has been demonstrated that a higher fiber intake is health beneficial, acting via anti-inflammatory and immune related pathways, which are mediated by the microbiota, but it is not clear to what extent the effects observed depend on the genetic background, which may be an important consideration for explaining health related effects of dietary patterns and interventions. It is the aim of this project to detect genetic variants related to responses of fiber-rich diets.

Methods

The proposed project will be carried out by building on an already planned human dietary intervention study in the context of another PhD thesis at DII (student: Erica Grant; MICROH DTU, focussing on the effects of short-term intervention of dietary fiber on the gut microbiota and the immune system, with a particular focus on the microbial mucus foraging). In the current DTU project, a novel and unique aspect will be whole genome analyses (MeGeno) of the healthy volunteers participating in the above interventional study. The genomic information of the healthy individuals will be analysed in relation to the data obtained in the intervention study. Specifically, the genomes analysed (and interesting variants retained) will be re-viewed (further scrutinized via pathway analyses such as KEGG…) and compared in terms of how different individuals would react to short-term dietary fiber intervention in terms of changes in the gut microbiota and inflammatory (CRP, miRNA, cytokines) changes. Immune cell profiling will also be carried out and associated to host genetics.

In a parallel study, samples collected during the cross-sectional ORISCAV-LUX study will be used to investigate the relation of fiber intake (e.g. highest vs. lowest quintile based on Food-Frequency Questionnaires), inflammatory markers (CRP, miRNA, cytokines etc.) and genetic background to verify whether findings from the intervention study can be found also in this population. A more targeted genetic analysis is envisioned, with variants chosen based on results of the intervention trial. This project will generate critical data sets and hypotheses to understand how genetic information can be potentially connected to various aspects of diet and the gut microbiota, making the present PhD project highly innovative, and is expected to result in additional high impact publications.

Project 1.4. - Modulation of cytokine pathways for allergy specific immunotherapy

Introduction

TNF-, IL-1 and IL-6 are well known to play a central role in the pathogenesis of multiple inflammatory diseases. They have also gained renewed interest as important players of T cell polarization, amongst others for the capacity to regulate the Th2 cell differentiation pathway. For example, IL-6 was shown to exert an important inhibitory role on Th2-cell differentiation, through a mechanism whereby DCs control Th2 immunity in a dominant repressive fashion (Mayer et al., Eur. J. Immunol., 2014). Impaired IL-6R function was recently associated to atopy, asthma, high IgE levels and systemic inflammation in two different patients with functionally impairing point mutations in the IL-6R (Spencer et al., J. Exp. Med., 2019). Finally yet importantly, very recent data from a clinical study performed by the Department of Infection and Immunity of LIH together with the Allergy-Immunology Service of CHL in Luxembourg (unpublished data; https://clinicaltrials.gov/ct2/show/NCT02931955) and from a preclinical murine allergy immunotherapy model (https://doi.org/10.22541/au.159557049.95398835) indicated that an early elevation of inflammatory cytokine levels is associated with successful immunotherapy outcome. Altogether, these observations suggest that new approaches for successful allergen immunotherapy and allergy prevention could be developed based on the modulation of the involved cytokine pathways.

Aim and Hypothesis

We will investigate defects in inflammatory cytokine pathways in genetically modified mouse models and will analyze the resulting modulations in the establishment of TH2 cell-mediated airway allergy and tolerance induction via allergen-specific immunotherapy treatment. During the last years, we have developed a highly characterized mouse model of allergic asthma to cat allergens, which can be used for preclinical evaluations of novel immunotherapeutic approaches. The adaptation of this model to mice with defects in the inflammatory cytokine signaling cascades will offer many opportunities to characterize the role of cytokine signaling on the development or resolution of allergy. We expect that mice with loss of function in these cytokine pathways will develop increased symptoms of allergy like airway hyper-reactivity, higher sIgE levels, and aggravated airway eosinophilia. Deep immune phenotyping and gene profiling of the key immune cells identified in the involved pathway will provide essential information to understand the mechanism driving the observed allergic phenotypes. On the opposite, we assume that amplifying the signal through these cytokines and their receptors could alleviate the symptoms of allergy and induce clinical tolerance.

Methods

Our team has already established the mouse model of allergic sensitization and all the techniques allowing the fine characterization of the animals through complementary read outs (airway hyper-reactivity, eosinophilia, Ig profiles, deep phenotyping of cells from lung, spleen or lymph nodes by flow cytometry or mass cytometry, and ex vivo functionality assays). We intend to isolate effector cells, T cells and Treg cells from control mice and mice with aggravated allergy for single cell sequencing and genomics/transcriptomics/epigenomics analyses. Part of the work will be devoted to developing the bioinformatics pipelines to process the big data collected in the project and to relate the findings of the preclinical model to human genomic data from existing patient cohorts.

Project 1.5. - Immunogenomics: Identification of genetic alterations causing immune dysfunctions of individual patients

Introduction

Next generation sequencing has revolutionized the entire field of basic and translational genomic research (Boycott et al., 2013). With enough coverage, the current sequencing technology with computational error-correction consensus-building approaches enabled an accurate and fast characterization of genetic mutations or variants on the entire human genome (Salk et al., 2018). Enormous progress has already been made in identifying causative/driver genetic variants of a variety of diseases, including cancer (Garraway et al., 2013), rare diseases (Chen et al., 2014) and immunemediated diseases in various organs (Christodoulou et al., 2013). However, the challenge remains in identifying the causative variants with a functional impact disturbing homeostasis out of the long list of potential candidate variants revealed.

Aim and Hypothesis

We hypothesize that many of the complex non-communicable diseases are at least partially caused by mutations or genetic variations of genes that are known to play a role in relevant immune functions and pathways. The aim of the project is to identify the causative genetic variants for the disease phenotypes, both subtle and overt, of the patients and healthy participants recruited and consented in the cohorts under the roof of the DTU.

Methods

We will employ whole-genome sequencing and whole-exome sequencing approaches to deeply characterize the genetic landscape of each consented patient or healthy participant recruited in selected cohorts of the current DTU. We will then constrain the variants identified by next generation sequencing approaches to genes of various known relevant immunological pathways (e.g. from the KEGG pathway collection), as well as to genes from a manually annotated immunomutation database under development by the partner group and the proteins interacting with those known immunological pathways. In this way, we can significantly narrow down4 the candidate list using prior knowledge in literature, immune function-related pathways and networks, and the expertise in immunology. The functional effects of the shortlisted novel candidate variants on the immune system will be investigated further in the corresponding projects.