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Development, maintenance and functions of Resident Macrophages.

Periodic Reporting for period 2 - ResidentMacroPhage (Development, maintenance and functions of Resident Macrophages.)

Reporting period: 2018-06-01 to 2019-11-30

Macrophages are professional phagocytic cells that orchestrate homeostatic and innate immune functions, via the scavenging of cells, debris, and pathogens, and the production of cytokines, both concurring to tissue homeostasis and repair. This project aims to investigate in vivo the development and functions of macrophages, which have considerable phenotypic and functional diversity depending on their tissue of residence and pathophysiological conditions. Because this diversity is not well understood, the functions of macrophages in vivo have not been well characterized. Most hematopoietic cells renew from hematopoietic stem cells (HSC), however recent evidence has shown that tissue ‘resident’ macrophages are generated from yolk sac progenitors, expand and differentiate within developing seeded tissues, and self-maintain in adults independently of HSC. In contrast to HSC-derived macrophages, resident macrophages proliferate locally in steady state and might self-renew. Thus, two lineages of macrophages coexist in most adult tissues. To critically assess the contribution of resident macrophages to tissue repair/regeneration, we aim to determine the developmental pathway of resident macrophages and the underlying molecular mechanisms that control their renewal and functions in vivo.
Given that the progressive replacement of heart resident macrophages with HSC-derived cells over time has been linked to age-associated cardiac fibrosis and deleterious outcomes following cardiac injury, a shift in ontogeny among tissue resident macrophages could contribute to age-related changes, such as impaired tissue repair. This work will significantly increase our knowledge of the differentiation, proliferation and function of resident macrophages in development and tissue repair and will open new venues of research into the role of self-renewing macrophages in regeneration and aging.
The overall objectives of the project are (i) to characterize the cellular and molecular events involved in resident macrophage differentiation from yolk sac progenitors, at the single-cell level in vivo, (ii) to identify critical transcription factors, as well as the relationship between cell cycle and differentiation in the resident macrophage lineage and, (iii) to characterize the specific function of resident macrophage during tissue repair/regeneration.
Our aim is to investigate the functions of tissue resident macrophages when homeostasis is perturbed (tissue repair, aging) and to decipher how resident macrophage commitment and differentiation is instructed from Hematopoietic Stem cell (HSC)-independent progenitors during fetal life. The team was able to recruit a postdoctoral fellow and a PhD student thanks to the ERC funding and collectively we have now made key advances on both fronts that will lead to publications in the next year.
First, we pioneered the study of resident macrophage maintenance during aging, evidencing for the first time that adult tissue resident macrophage density is reduced at least two-fold in most aged tissues and this is solely due to the specific loss of embryo-derived HSC-independent macrophages. This loss is not due to macrophage proliferative exhaustion but rather is induced by sustained inflammation. Impaired inflammation sensing during aging did not protect from the low chronic inflammation observed in aged animals but was sufficient to maintain resident macrophage density at younger levels. This had physiological implications as key features of aging contributing to tissue dysfunction were not observed in the liver of animals that have maintained their embryo-derived resident macrophages during aging. Importantly, contrary to what is observed during acute inflammation, loss of embryo-derived macrophages during aging was not accompanied by a compensatory increase of HSC-derived macrophages (Saenz Coronilla, Ade et al., manuscript submitted).
Secondly, we are dissecting the contribution to fetal hematopoiesis of yolk sac progenitors that emerge in the embryo before HSC generation and that are the main source of adult tissue resident macrophages. We have dissected yolk sac progenitor heterogeneity in their niche of emergence (yolk sac) using high-parameter flow cytometry, imaging, functional in vitro tests and single cell RNA sequencing (scRNAseq) (Iturri et al., manuscript in preparation). In collaboration with Ana Cumano’s team (Institut Pasteur), we have characterized their contribution to fetal erythropoiesis (Soares Silva et al., manuscript in preparation) and confirmed their lack of contribution to lymphocyte lineages (El Said et al., manuscript in preparation).
Progress beyond the state
One of the current challenges in better understanding the biology of yolk sac progenitors and their contribution to mature differentiated blood and tissue cells is the lack so far of specific tools to target or label them, as most cell surface markers and transcription factors involved in their generation are partially shared with HSC and HSC-derived progenitors. To circumvent this, we are combining genetic pulse labelling with single cell transcriptomic approaches. To improve the current limitations of well-known genetic mouse models of key transcription factor deficiencies, we are combining them with inducible genetic labelling systems that will allows us to ascribe phenotypes to specific fetal hematopoietic progenitors waves. Finally, to gain functional knowledge on resident macrophages and their progenitors, we are establishing new mouse models combining inducible Cre systems with a wide range of reporter strains that will be analysed using not only quantitative high-parameter flow cytometry but also in situ immunofluorescence approaches. We have thus far evidenced that yolk sac progenitors actually encompass a heterogeneous population of bipotent and committed progenitors and we have challenged the current paradigm that yolk sac-derived HSC-independent macrophages are systematically replaced by HSC-derived cells in adult tissues.

Expected results:
We are dissecting the differentiation potential of yolk sac-derived progenitors in the fetal liver hematopoietic niche, which they share with HSC. We are currently building up a time and space (fetal liver, blood, peripheral tissues) map of the contribution of yolk sac-derived progenitors to myeloid lineages (macrophages, monocytes, neutrophils and mast cells) using genetic pulse labelling of yolk sac progenitors and HSCs. Finally, we are building an unbiased EMP differentiation tree from its two niches and at different timepoints using retrospective lineage tracing by performing scRNAseq from pulsed labelled yolk sac progenitors and fate-mapped HSC counterparts. This work will allow us to dissect macrophage commitment and differentiation during embryonic development but it will also allow us to better understand the specificities and requirement of yolk sac progenitors when compared to HSC-derived hematopoiesis. The datasets generated will allows us to characterize the transcription factors and gene networks underlying progenitor commitment. We will use a combination of high-parameter flow cytometry analysis of genetic mutants, in vitro potency assays (colony forming assays), live imaging of whole embryo and whole organ culture (yolk sac versus fetal liver) and single-cell live-imaging of progenitor subsets in microfluidic devices to functionally validate the results thus far obtained. This will generate an important resource for the community and lead to one or two manuscripts until the end of the project.

We are currently dissecting the ontogeny and dynamics of macrophages during tissue repair and fibrosis. Liver resident macrophages (also known as Kupffer cells) are relatively well characterized but their contribution to liver regeneration and fibrosis is still not completely understood, in particular in comparison to HSC-derived macrophages. In the skin, we have identified a novel population of yolk sac-derived macrophages that do not correspond to either perivascular or peri-nerve macrophages. We are further characterizing this population using fate mapping approaches, shielded bone marrow chimeras and scRNAseq (10x genomics) and investigating their contribution to skin fibrosis in two complementary models. We expect to characterize how HSC-independent resident macrophage populations are maintained during steady-state and tissue repair and identify their function(s). This work will lead to a manuscript describing the contribution of resident macrophages to liver tissue repair and another characterizing HSC-independent skin macrophages.