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Development of the immune system – IMMUNODEV

Development of the Immune System

it is still unclear how lymph nodes and lymphatics are initiated at specific locations. Based on my results and on pilot data, nerve fibers and blood vessel bifurcations could play a role. My overall objective is to characterize all players involved in development of the immune system, specifically in relation to the formation of lymph nodes and lymphatic vasculature.

My overall objective is to characterize all players involved in development of the immune system, specifically in relation to the formation of lymph nodes and lymphatic vasculature.

Lymph nodes and lymphatic vasculature are an essential part of the adaptive immune system. Properly placed lymphatic vessels and lymph nodes are crucial for an adequate and fast immune response. It is therefore not surprising that both lymphatic vessels and lymph nodes are formed in a coordinated fashion, as lymphatic vessels and lymph nodes form within the same time frame, while sharing essential differentiation signals.<br />Lymph nodes are formed during embryogenesis, and the first steps in this process involve the interaction of mesenchymal organizer cells with type 3 innate lymphoid cell (ILC3) under the influence of retinoic acid at specific locations throughout the body. Interestingly, the source for retinoic acid, or the location where vitamin A is converted into retinoic acid near the embryonic lymph nodes or jugular lymph sacs is not known yet. The finding that nerve fibers are always located near the sites of lymph node and jugular lymph sac formation, and that they express high levels of the retinoic acid synthesizing enzyme implies that they might be involved in development of the lymph nodes and the lymphatic vasculature. It is very interesting to speculate that neurons are involved in the formation of the immune system, and it has been shown before that they play a role in regulation of gut immunity and in the release of stem cells in the bone marrow. <br /><br />My specific key objectives are:<br />1. What determines the location of lymph node initiation?<br />2. What determines the location of lymphatic vasculature initiation?<br />3. How are lymphatic endothelial cells attracted towards and connect to embryonic lymph nodes?

It is fascinating that multiple cell types at a specific location within an embryo are involved in the initiation of lymphoid tissues. The complex interaction between mesenchymal organizer and hematopoietic ILC3s has been described extensively. However, the notion that neurons could be involved in this process is very new. This also poses a difficult challenge, since it is not possible to study mammalian models that lack neurons. Moreover, there are no specific neuronal drivers available, since many genes are also expressed in other cell types. Finally, peripheral nerve bundles in mouse models with defective neuronal outgrowth like the Frizzled-/- model have slightly changed locations which are difficult to quantify. Therefore, it is essential to generate
high resolution and accurate 3D models, in which the location of the nerve bundles in relation to the lymph nodes can be mapped. The development of the light sheet microscopy techniques allows the acquisition and rendering of relative large 3D fluorescently stained tissues, like the E13.5 and E14.5 mouse embryos, in which the lymph nodes are present. I have successfully imaged whole mount stained E13.5 and E14.5 mouse embryos during a pilot study. In this project, I wish to continue on this subject and study in detail the location of the involved cell types in 3D, using a light sheet microscope. The project will use different reporter and knock out mouse models to establish cellular and molecular mechanisms involved. The interdisciplinary approach of this project will substantially contribute to the understanding how neurons are involved, either during normal embryonic immune system development, but likely also during chronic inflammation when ectopic structures are formed.

Collaborations with the groups of prof.dr.Kiefer (MaxPlanck Institute Münster), prof.dr.Nibbs (University Glasgow) and prof.dr.Stumm (Jena University) allowed me to contribute on manuscripts and which have been submitted and/or published. We are now in the process of finishing some of the projects for publication.
We could show that the chemokine is expressed near and in the lymph node anlagen and important for the amount of Lymphoid Tissue Inducer cells within the lymph node anlage. We have generated tissue specific knock-out mice to study the role for the chemokine and its receptor is more detail and could show that the complete knock-out for the chemokine results in smaller lymph node anlagen. Also, the knockout for the receptor on the Lymphoid Tissue Inducer cells shows a reduction, but not as strong as the chemokine knockout. The deletion of the chemokine from endothelial cells did not show any difference, therefore we can conclude that the role of the chemokine of the Lymphoid Tissue Inducer cells is probably from within the lymph node anlagen. We have acquired a mouse model to delete the chemokine from these cells within the lymph node anlagen and plan to finish the experiments before the end of the project.
We are studying the ontogeny of the Lymphoid Tissue Inducer cells by using 2 lineage tracing models, in part setup by a colleboration with prof.dr.Stumm. With these models, we could show the specific contribution from one of the two embryonic regions from which hematopoietic cells arise. Furthermore, we could now show that the differentiation towards the mature Lymphoid Tissue Inducer cells occurs most likely in the periphery and not the fetal liver, answering a long standing question.

Due to our expertise on the lightsheet microscopy we collaborate with several national and international groups. This has gained us succesfull collaborative grants (ANR-PRC and ARC-INCA Pair) to study whole mount immune organs or cancers. Moreover, we have contributed to papers (Journal of Immunology, 2018; Glia 2018; Journal Cell Science 2018) within these collaborations.

- 3 manuscript published as collaborator
- 1 manuscript submitted for publication as collaborator
- 2 review manuscripts on ILC3s
- ANR PRCI «ATTRACT« 2017, due to collaboration on cell migration
- ARC/INCA Pair as collaborator
- ANR PRC as collaborator
- Many invited seminars

During an infection antigens are transported from the infected area through the lymphatic vasculature towards the nearest lymph node. Within the highly organized lymph node, an adaptive immune response is mounted in the appropriate compartment to be able to combat the infection. Hence, properly placed lymphatic vessels and lymph nodes are crucial for an adequate and fast immune response.
Lymph nodes are formed within the embryo, and the first steps in this process involve the interaction of mesenchymal organizer cells with type 3 innate lymphoid cells at specific locations throughout the embryo. Also, development of the lymphatic vasculature is initiated at specific locations within the embryo. At these locations, blood vessel endothelial cells differentiate into lymphatic endothelial cells, which will bud of from the blood vessels and form larger lymphatic structures nearby. Subsequently, superficial lymphatic vessels are formed and sprout throughout the body and attach to lymph nodes. Embryonic development of the lymphatic vasculature occurs in a coordinated fashion with formation of lymph nodes. Although they initially form independently, shortly afterwards they are connected to each other. The cellular and molecular mechanisms that are involved in the attraction and connection of lymphatic vasculature to embryonic lymph nodes are unknown.
My overall objective is to characterize all players involved in development of the immune system, specifically in relation to the formation of lymph nodes and lymphatic vasculature. What determines the location of initiation and what occurs at these sites? To be able to study these regions, 3D reconstructions will be generated, in which the anatomical relation between the key players will be examined. Specifically, the type 3 innate lymphoid cells, lymphatic vasculature, blood vessels and nerve bundles will be stained and analyzed. Reporter and mutant mouse models for these key players will be used to investigate the molecular mechanisms involved in the development of the immune system.
This project aims to provide a basic understanding on the mechanisms that regulate formation of the lymphatic vasculature and lymph nodes at specific locations within the embryo. This knowledge could be extremely useful in situations when the formation of ectopic lymph nodes and lymphatics is needed, like in lymphedema, or needs to be prevented, like in chronic inflammations. Knowledge on factors involved in lymph node development, such as retinoic acid, could aid the generation of ectopic development in lymph nodes in areas where they are needed. For example, in breast cancer patients, lymph nodes are often resected resulting in affected lymphatic drainage in about 30-40% of the cases. Regrowth of lymph nodes, together with their associated lymphatic vasculature, will substantially improve quality of live in these patients. Also, ectopic lymph nodes are formed during chronic inflammation, such as rheumatoid arthritis, and in most cases are detrimental for the outcome of the disease. When the factors are known that govern lymph node formation, blocking their function during chronic inflammation but before ectopic lymph node formation will aid the outcome of the disease.

Project coordinator

Monsieur Serge VAN DE PAVERT (Institut National de la Santé et de la Recherche Médicale délégation PACA et Corse_Centre d'Immunologie de Marseille Luminy)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM PACA et Corse_CIML Institut National de la Santé et de la Recherche Médicale délégation PACA et Corse_Centre d'Immunologie de Marseille Luminy

Help of the ANR 325,000 euros
Beginning and duration of the scientific project: June 2016 - 48 Months

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