Periodic Reporting for period 4 - ResidentMacroPhage (Development, maintenance and functions of Resident Macrophages.)
Reporting period: 2021-06-01 to 2023-05-31
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 aimed 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. In contrast to hematopoietic stem cells (HSC)-derived macrophages, Resident macrophages are generated from yolk sac progenitors, expand and differentiate within developing seeded tissues, and self-maintain in adults independently of HSC. Thus, two lineages of macrophages coexist in most adult tissues. The overall objectives of the project were to characterize (i) the developmental trajectory of yolk sac progenitors, at the single-cell level in vivo, (ii) how macrophage networks are maintained and, (iii) the specific function of resident macrophage during tissue repair/regeneration.
First, we elucidated the heterogeneity of HSC-independent progenitors that emerge from the yolk sac and characterized their niche-specific developmental pathways when committing and differentiating into erythrocytes, megakaryocytes, mast cells, neutrophils and macrophages. Importantly, we found that HSC-independent erythromyeloid progenitors (EMP) sustain the production of red blood and myeloid cells until birth, with little to no input from HSC. Second, we demonstrated that low grade chronic inflammation was responsible for the loss of HSC-independent macrophages over time without hampering their proliferative capacities. And lastly, we have characterized macrophage ontogeny and functions during placental organogenesis, skin wound healing and after myocardial infarction.
The results from this project have laid the foundation of a new framework to understand how the immune system contributes to the developmental origins of health and disease, by focusing on transient hematopoietic progenitors and their long-lived progeny, the tissue resident macrophages. Finally, uncovering the developmental pathways and molecular mechanisms involved in these fetal progenitors will contribute to improve of understanding of pediatric disorders.
First, we elucidated the heterogeneity of HSC-independent progenitors that emerge from the yolk sac and characterized their niche-specific developmental pathways when committing and differentiating into erythrocytes, megakaryocytes, mast cells, neutrophils and macrophages. Importantly, we found that HSC-independent erythromyeloid progenitors (EMP) sustain the production of red blood and myeloid cells until birth, with little to no input from HSC. Second, we demonstrated that low grade chronic inflammation was responsible for the loss of HSC-independent macrophages over time without hampering their proliferative capacities. And lastly, we have characterized macrophage ontogeny and functions during placental organogenesis, skin wound healing and after myocardial infarction.
The results from this project have laid the foundation of a new framework to understand how the immune system contributes to the developmental origins of health and disease, by focusing on transient hematopoietic progenitors and their long-lived progeny, the tissue resident macrophages. Finally, uncovering the developmental pathways and molecular mechanisms involved in these fetal progenitors will contribute to improve of understanding of pediatric disorders.
During the project, we developed two main axis of research, the first focused on characterizing the specific functions of resident macrophage during development, tissue repair and aging. First, we characterized the ontogeny of embryo-derived macrophages found in the placenta (Hofbauer cells) and demonstrated their implication in placental organogenesis and in its vascularization in particular. We also established tissue repair models (liver failure, wound healing, myocardial infarction) where we implemented macrophage depletion and single cell transcriptomic approaches in order to unravel the role of long-lived fetal macrophages in preventing tissue fibrosis and promoting vascular remodeling after injury. We undertook the risky project of investigating macrophage maintenance during aging and we demonstrated that chronic inflammation induces macrophage loss with age and tissue dysfunction. Further, loss of long-lived macrophages does not trigger their replacement by new recruited macrophages. This has 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.
Our second research axis focused on characterizing the cellular and molecular events involved in resident macrophage differentiation from yolk sac (YS) progenitors, at the single-cell level in vivo. While initially focused on macrophages, we expanded our field of study since we demonstrated that these progenitors sustain the production of red blood cells, platelets, mast cells and neutrophils until birth. We then elucidated the contribution to fetal hematopoiesis of these YS progenitors. We have dissected yolk sac progenitor heterogeneity in their niche of emergence (YS) using high-parameter flow cytometry, imaging, functional in vitro tests and single cell RNA sequencing. Finally, we uncovered that cell fate commitment from fetal hematopoietic progenitors is differently regulated in time and space, leading to a tightly controlled production of mature cells from different progenitor waves and from different niches. Furthermore, we identified several mechanisms underlying how transient progenitors outcompete HSCs during development, particularly how these progenitors appeared poised for differentiation into red blood cell although they had already committed to other lineages.
Our second research axis focused on characterizing the cellular and molecular events involved in resident macrophage differentiation from yolk sac (YS) progenitors, at the single-cell level in vivo. While initially focused on macrophages, we expanded our field of study since we demonstrated that these progenitors sustain the production of red blood cells, platelets, mast cells and neutrophils until birth. We then elucidated the contribution to fetal hematopoiesis of these YS progenitors. We have dissected yolk sac progenitor heterogeneity in their niche of emergence (YS) using high-parameter flow cytometry, imaging, functional in vitro tests and single cell RNA sequencing. Finally, we uncovered that cell fate commitment from fetal hematopoietic progenitors is differently regulated in time and space, leading to a tightly controlled production of mature cells from different progenitor waves and from different niches. Furthermore, we identified several mechanisms underlying how transient progenitors outcompete HSCs during development, particularly how these progenitors appeared poised for differentiation into red blood cell although they had already committed to other lineages.
One of the current challenges in better understanding the biology of yolk sac progenitors (EMP) and their contribution to mature cells is the lack so far of specific tools to target or label them, as most cell surface markers and transcription factors are partially shared with HSC and HSC-derived progenitors. To circumvent this, we combined genetic pulse labelling of yolk sac-derived progenitors in three complementary fate mapping models with single cell transcriptomic approaches. The datasets generated allowed us to characterize the time- and niche-specific developmental trajectories underlying progenitor commitment by using a combination of genetic mutants, in vitro potency assays (colony forming assays) and in vivo functional assays (transplantation). We identified two temporally distinct developmental trajectories from EMP and demonstrated that EMP are the predominant source of mature erythroid and myeloid cells until birth. In the fetal liver, EMP efficiently outcompete HSC progeny, which fails to generate erythroid progenitors in vivo. Collectively, our work thereby establishes a developmentally-restricted privilege for erythro-myeloid differentiation from EMP compared to HSC and we uncovered a dichotomy in the allocation of fetal liver EMP and HSC to myeloid progenitor subsets, both in timing and lineage bias. This provides a framework for future studies of fetal hematopoiesis. This work has generated an important resource for the community and has led to four manuscripts, all available openly for the community.
In regards to resident macrophages, the long-lived progeny of EMP, we dissected the ontogeny and dynamics of macrophages during placenta formation, tissue repair and aging. EMP are the major source of embryo-derived placental macrophages (or hofbauer cells, HBC), which play a key role in labyrinth angiogenesis and/or remodeling. This work provides groundwork for future investigation into the relationship between HBC ontogeny and function in placenta pathophysiology. Our work has contributed to the understanding of macrophage heterogeneity, and associated macrophage ontogeny with distinct functions in health and disease of the cardiovascular system. Finally, we showed that resident macrophage numbers dwindles with age in most tissues, without compensation from HSC, due to chronic inflammation-induced cell death. Attenuating inflammation sensing during ageing prevents age-induce macrophage loss and improves hallmarks of liver ageing.
In regards to resident macrophages, the long-lived progeny of EMP, we dissected the ontogeny and dynamics of macrophages during placenta formation, tissue repair and aging. EMP are the major source of embryo-derived placental macrophages (or hofbauer cells, HBC), which play a key role in labyrinth angiogenesis and/or remodeling. This work provides groundwork for future investigation into the relationship between HBC ontogeny and function in placenta pathophysiology. Our work has contributed to the understanding of macrophage heterogeneity, and associated macrophage ontogeny with distinct functions in health and disease of the cardiovascular system. Finally, we showed that resident macrophage numbers dwindles with age in most tissues, without compensation from HSC, due to chronic inflammation-induced cell death. Attenuating inflammation sensing during ageing prevents age-induce macrophage loss and improves hallmarks of liver ageing.