Formation of Tertiary Lymphoid Structures (TLS) is a recurring consequence of chronic tissue inflammation and autoimmunity, as well as several solid tumours. TLS originate in various tissues from infiltrating cells attracted by local signalling cascades. These structures can enhance local immune response by, similarly to Secondary Lymphoid Organs (SLO), functioning as spots for lymphocyte activation upon antigen recognition. However, the cellular interactions driving TLS emergence and function across tissues are not fully known. This proposal aims to unravel and characterise the cell-cell interactions involved in the formation and maintenance of TLS, in particular their tissue specificity and relationship to autoimmune diseases. A comprehensive understanding of intercellular interactions will be obtained from publicly available and newly generated single-cell RNA-sequencing (scRNA-seq) datasets from TLS-containing tissues of autoimmunity patients. Molecules involved in key signalling interactions will be spatially resolved by assessing their tissue expression using spatial transcriptomics. Then, using mouse models where TLS triggering can be controlled, scRNA-seq data will be obtained to outline a time course of TLS formation in different tissues, revealing the temporal role of the discovered interactions and their specific function in each tissue and when compared with SLOs. Finally, regulation and downstream effects of ligands and receptors will be unraveled with multiome (RNA+open chromatin) sequencing using human tonsil organoids. The observed effects will be generalised to other tissues using machine learning and deep learning models, resulting in a tissue-specific reference of signalling and regulatory genes controlling TLS biogenesis. Overall, this project aspires to provide a detailed look into TLS biology, while defining the bases for their therapy-oriented manipulation.

Neuromuscular disorders (NMDs) are a collection of pathologies that affect the skeletal muscle (SkM) function and lead to reduced mobility, breathing issues and death. Most NMDs are incurable and existing therapies are generally ineffective. The major contributor for the lack of NMD therapies is the absence of human NMD research models, applicable for the screening and validation of therapeutic candidates. Our goal for this project is to develop an in vitro platform containing highly biomimetic and functional SkM analogues, where NMDs can be modelled with the aim of testing drug candidates, in a reliable, high-throughput (HT), and quick manner. The platform will consist of 96 individual 3D tissue units, generated from the myogenic differentiation of human induced pluripotent stem cells (hiPSCs). To induce biomimetic and mature tissue formation, the platform will combine a unique topographical design with a light-stimulation system for optogenetic control of SkM contraction. Using image-based assays and computational algorithms, we will be able to automatically extract and quantify the SkM phenotypical (e.g. intracellular structure) and functional (e.g. contraction speed/force) parameters. To show NMD modelling ability within the platform, we will knockout BIN1, a gene whose mutated form is involved in the onset of centronuclear myopathy (CNM), a fatal and incurable NMD. The validity of the NMD model as a drug discovery platform will be demonstrated by testing compounds with known therapeutic activity. This proof of concept project will lay the ground work for a spin-off company devoted to HT drug screening for NMDs. The successful development of this platform will provide an invaluable tool for pharmaceutical research, allowing accurate, convenient and cost-effective identification of novel drugs and therapeutic targets for NMDs and, ultimately contributing to the acceleration of novel therapies for NMD patients.

Background: The study of protozoan pathogens has been extensively explored often motivated to find suitable targets for new intervention strategies. However these studies have been mostly limited to those life-cycle stages that can be cultivated in vitro. Using a mouse model of African trypanosomiasis, we have recently discovered that the adipose tissue (fat) is a major reservoir for the extracellular protozoan Trypanosoma brucei and that, within this environment, parasites become phenotypically different from those in the blood. Our study exposed novel biology of the T. brucei life cycle, yet it remains unknown how parasites adapt to the fat and how parasite fat tropism affects disease. Our first aim is to determine the molecular and cellular mechanisms underlying T. brucei fat tropism. We will perform a genetic screen in mice to identify key parasite genes required for establishing and maintaining chronic infection in the fat. Together with the information of the transcriptome and proteome, we will identify the mechanistic steps underlying parasite tissue-adaptation. Our second aim is to identify the consequences of T. brucei fat tropism for the host and the importance for disease. We will first investigate if parasites can egress from the fat. We will also determine if parasites induce lipid breakdown in the host, leading to loss of fat mass. Finally, we will measure the impact of fat tropism in general traits of disease, including host survival and transmission potential. Impact: This project represents a completely novel research avenue built on recent work from my laboratory. By uncovering fundamental aspects of the biology of T. brucei, we will also improve the understanding of clinically relevant features of African trypanosomiasis, including relapses and weight loss. In addition, since parasite fat tropism has also been observed in malaria and Chagas’ disease, our findings will help elucidate disease mechanisms relevant to other infectious diseases.

Adult stem cells (SCs) sustain tissue renewal and repair throughout life. The SC niche is fundamental in the regulation of SC function and an important contributor to SC decline in aging. While alterations in the tissue’s immune environment are emerging as important contributors to impairments found in aged organs, their contribution to SC dysfunction in aging is unknown. The skeletal muscle (SkM) is a paradigmatic model to study age-related loss of repair capacity. Muscle stem cell (MuSC) function during regeneration requires plasticity in transit between states of quiescence, activation and differentiation. MuSC functional impairments in aging result from changes in the extrinsic cues that govern their behavior, but also cell intrinsic alterations, including senescence and defects in lineage commitment. However, we still have a limited understanding of how changes in the environment manifest as SC intrinsic defects. Our previous work indicates that changes in immune signaling are important drivers of MuSC dysfunction and regenerative decline in aging. Here, we propose to identify the contribution of specific immune populations and signals to changes in regenerative capacity and MuSC activity in aging (Aim 1). We hypothesize that the immune environment is an essential regulator of MuSC plasticity and lineage commitment under regenerative pressure, and immune alterations underlie defects in MuSC lineage fidelity in aging. We propose to map the trajectories of MuSCs diverging from the myogenic lineage and uncover the changes in epigenetic landscape that underlie the loss of lineage fidelity associated with immune aging, identifying transcriptional regulators of MuSC fate (Aim 2). The knowledge generated on the mechanisms linking immune aging and MuSC dysfunction will be tested for the conservation in human SkM and will be applied to improve the success of MuSC-based therapies in aging (Aim 3).

The mammalian gut is an exquisite system to study ecology and evolution of microbes, and these processes are key for host-microbiome homeostasis. How microbiome diversity is maintained or lost is a critical question underlying the proper balance of this duet. Yet, our knowledge of the eco-evolutionary mechanisms structuring microbiomes is still in its infancy. Here, we seek to identify dominant modes of natural selection and host factors that modulate the evolution of their microbes. By leveraging knowledge on gene functions in specific strains and the power of mouse genetics and husbandry, we will unravel how natural selection operates to shape diversity in the bacteria that inhabit the guts of healthy and sick hosts. Microbiome evolution will be studied in several mouse models of disease with a focus on Escherichia coli as a pathobiont model, for which a deeper understanding of molecular mechanisms in health vs disease can be reached. Using long-term experimental evolution in vivo, high-throughput sequencing and theoretical modelling we will quantify the relative roles of directional, diversifying and fluctuating selection in gut evolution. We posit that resource competition drives the dominant selection mode in the healthy gut and that strong fluctuations in the environment, due to phage-bacteria co-evolution and/or due to hostmicrobe interactions, drive the selection mode in the gut of diseased hosts. We will further test the hypothesis that fluctuating selection leads to an Anna Karenina effect whereby the microbiomes of unhealthy individuals are much more distinct between one another than those of heathy ones. EvoInHi seeks to find the first empirical evidence that the predictability of evolution is higher in health than in disease, which will have a profound impact on understanding bacteria diversity and rates of specialization and how these can be used to modulate host health.