In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

Lymphatic Endothelial Cells Are Essential Components of the Subcapsular Sinus Macrophage Niche.

In lymph nodes, subcapsular sinus macrophages (SSMs) form an immunological barrier that monitors lymph drained from peripheral tissues. Upon infection, SSMs activate B and natural killer T (NKT) cells while secreting inflammatory mediators. Here, we investigated the mechanisms regulating development and homeostasis of SSMs. Embryonic SSMs originated from yolk sac hematopoiesis and were replaced by a postnatal wave of bone marrow (BM)-derived monocytes that proliferated to establish the adult SSM network. The SSM network self-maintained by proliferation with minimal BM contribution. Upon pathogen-induced transient deletion, BM-derived cells contributed to restoring the SSM network. Lymphatic endothelial cells (LECs) were the main source of CSF-1 within the lymph node and conditional deletion of Csf1 in adult LECs decreased the network of SSMs and medullary sinus macrophages (MSMs). Thus, SSMs have a dual hematopoietic origin, and LECs are essential to the niche supporting these macrophages.

Hemogenic Endothelial Fate Mapping Reveals Dual Developmental Origin of Mast Cells.

Hematopoiesis occurs in distinct waves. "Definitive" hematopoietic stem cells (HSCs) with the potential for all blood lineages emerge in the aorta-gonado-mesonephros, while "primitive" progenitors, whose potential is thought to be limited to erythrocytes, megakaryocytes, and macrophages, arise earlier in the yolk sac (YS). Here, we questioned whether other YS lineages exist that have not been identified, partially owing to limitations of current lineage tracing models. We established the use of Cdh5-CreERT2 for hematopoietic fate mapping, which revealed the YS origin of mast cells (MCs). YS-derived MCs were replaced by definitive MCs, which maintained themselves independently from the bone marrow in the adult. Replacement occurred with tissue-specific kinetics. MCs in the embryonic skin, but not other organs, remained largely YS derived prenatally and were phenotypically and transcriptomically distinct from definite adult MCs. We conclude that within myeloid lineages, dual hematopoietic origin is shared between macrophages and MCs.

Mast cell ontogeny: From fetal development to life-long health and disease.

Mast cells (MCs) are evolutionarily ancient innate immune cells with important roles in protective immunity against bacteria, parasites, and venomous animals. They can be found in most organs of the body, where they also contribute to normal tissue functioning, for example by engaging in crosstalk with nerves. Despite this, they are most widely known for their detrimental roles in allergy, anaphylaxis, and atopic disease. Just like macrophages, mast cells were conventionally thought to originate from the bone marrow. However, they are already present in fetal tissues before the onset of bone marrow hematopoiesis, questioning this dogma. In recent years, our view of myeloid cell ontogeny has been revised. We now know that the first mast cells originate from progenitors made in the extra-embryonic yolk sac, and later get supplemented with mast cells produced from subsequent waves of hematopoiesis. In most connective tissues, sizeable populations of fetal-derived mast cells persist into adulthood, where they self-maintain largely independently from the bone marrow. These developmental origins are highly reminiscent of macrophages, which are known to have critical functions in development. Mast cells too may thus support healthy development. Their fetal origins and longevity also make mast cells susceptible to genetic and environmental perturbations, which may render them pathological. Here, we review our current understanding of mast cell biology from a developmental perspective. We first summarize how mast cell populations are established from distinct hematopoietic progenitor waves, and how they are subsequently maintained throughout life. We then discuss what functions mast cells may normally have at early life stages, and how they may be co-opted to cause, worsen, or increase susceptibility to disease.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

T Cell Zone Resident Macrophages Silently Dispose of Apoptotic Cells in the Lymph Node.

In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1+ MERTK+ cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis.

Macrophage-fibroblast circuits in the spleen.

Macrophages are an integral part of all organs in the body, where they contribute to immune surveillance, protection, and tissue-specific homeostatic functions. This is facilitated by so-called niches composed of macrophages and their surrounding stroma. These niches structurally anchor macrophages and provide them with survival factors and tissue-specific signals that imprint their functional identity. In turn, macrophages ensure appropriate functioning of the niches they reside in. Macrophages thus form reciprocal, mutually beneficial circuits with their cellular niches. In this review, we explore how this concept applies to the spleen, a large secondary lymphoid organ whose primary functions are to filter the blood and regulate immunity. We first outline the splenic micro-anatomy, the different populations of splenic fibroblasts and macrophages and their respective contribution to protection of and key physiological processes occurring in the spleen. We then discuss firmly established and potential cellular circuits formed by splenic macrophages and fibroblasts, with an emphasis on the molecular cues underlying their crosstalk and their relevance to splenic functionality. Lastly, we conclude by considering how these macrophage-fibroblast circuits might be impaired by aging, and how understanding these changes might help identify novel therapeutic avenues with the potential of restoring splenic functions in the elderly.

Remodeling of reactive lymph nodes: Dynamics of stromal cells and underlying chemokine signaling.

Lymph nodes (LNs) are secondary immune organs dispersed throughout the body. They are primarily composed of lymphocytes, "transient passengers" that are only present for a few hours. During this time, they extensively interact with a meshwork of stromal cells. Although these cells constitute less than 5% of all LN cells, they are integral to LN function: Stromal cells create a three-dimensional network that provides a rigid backbone for the transport of lymph and generates "roads" for lymphocyte migration. Beyond structural support, the LN stroma also produces survival signals for lymphocytes and provides nutrients, soluble factors, antigens, and immune cells collectively required for immune surveillance and the generation of adaptive immune responses. A unique feature of LNs is their ability to considerably and rapidly change size: the volume and cellularity of inflamed LNs can increase up to 20-fold before returning to homeostatic levels. This cycle will be repeated many times during life and is accommodated by stromal cells. The dynamics underlying this dramatic remodeling are subject of this review. We will first introduce the main types of LN stromal cells and explain their known functions. We will then discuss how these cells enable LN growth during immune responses, with a particular focus on underlying cellular mechanisms and molecular cues. Similarly, we will elaborate on stromal dynamics mediating the return to LN homeostasis, a process that is mechanistically much less understood than LN expansion.

SIRT1 regulates macrophage self-renewal.

Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.

Lymph Node Stroma Dynamics and Approaches for Their Visualization.

Lymphoid stromal cells are best known as the architectural cells of lymphoid organs. For decades, they have been considered as inert elements of the immune system but this view has changed dramatically in recent years, when it was discovered that they are endowed with critical immunoregulatory functions. It is now accepted that without them, the adaptive immune response would be compromised, if not abrogated entirely. Here, we review the function of the major lymphoid stromal cell types; the way they remodel upon inflammation; discuss the available tools to track their behavior; and introduce several methodological approaches that we believe will help improving our knowledge of these pivotal cell types.

Lymph node macrophages: Scavengers, immune sentinels and trophic effectors.

Lymph nodes (LN) are secondary lymphoid organs dispersed throughout the body that filter lymph and assist the immune system in mounting immune responses. These functions are supported by a complex stromal microarchitecture composed of mesenchymal and vascular elements. Different subsets of macrophages (MΦ) reside in the LN and are endowed with immune and trophic functions. Here we review these different subsets with particular emphasis on the recently described T cell zone MΦ. We also address the potential crosstalk between LN stromal cells and MΦ, proposing that the former constitute niches for the latter by supplying factors required for their specification, survival and turnover. In turn, MΦ could inform their stromal partners about the immune status of the LN and orchestrate the remodelling of its microanatomy during immune responses.

Tissue macrophage identity and self-renewal.

Macrophages are cellular components of the innate immune system that reside in virtually all tissues and contribute to immunity, repair, and homeostasis. The traditional view that all tissue-resident macrophages derive from the bone marrow through circulating monocyte intermediates has dramatically shifted recently with the observation that macrophages from embryonic progenitors can persist into adulthood and self-maintain by local proliferation. In several tissues, however, monocytes also contribute to the resident macrophage population, on which the local environment can impose tissue-specific macrophage functions. These observations have raised important questions: What determines resident macrophage identity and function, ontogeny or environment? How is macrophage proliferation regulated? In this review, we summarize the current knowledge about the identity, proliferation, and turnover of tissue-resident macrophages and how they differ from freshly recruited short-lived monocyte-derived cells. We examine whether macrophage proliferation can be qualified as self-renewal of mature differentiated cells and whether the concepts and molecular pathways are comparable to self-renewal mechanisms in stem cells. Finally, we discuss how improved understanding of macrophage identity and self-renewal could be exploited for therapeutic intervention of macrophage-mediated pathologies by selectively targeting freshly recruited or resident macrophages.

The Innate Immune Response in Myocardial Infarction, Repair, and Regeneration.

Following myocardial infarction (MI), resident innate immune cells such as macrophages, innate lymphoid cells, and mast cells rapidly coordinate their function to contain inflammation by removing dying cells and promoting cardiomyocyte replenishment. To sustain local tissue repair functions, hematopoietic progenitors are mobilized from the bone marrow to the spleen to generate subsequent myeloid cells such as monocytes and neutrophils, which are rapidly recruited at the site of MI. A finely tuned balance between local adaptation and recruitment controls the overall outcome of the cardiac tissue regeneration versus repair and scar formation.In this chapter, the (potential) roles of the innate immune system residing in the heart are discussed in the context of recent findings about macrophage ontogeny and their homeostasis with circulating monocytes during cardiac tissue growth and after myocardial infarction. Their interactions with other members of the innate immune system are also discussed with a particular emphasis on the potential involvement of mast cells and innate lymphoid cells during MI, largely underestimated until recently. Understanding the development and the functions of the different protagonists responding to MI as well as their potential cross talk could help design new strategies for regenerative medicine intervention.

Notch controls the magnitude of T helper cell responses by promoting cellular longevity.

Generation of effective immune responses requires expansion of rare antigen-specific CD4(+) T cells. The magnitude of the responding population is ultimately determined by proliferation and survival. Both processes are tightly controlled to limit responses to innocuous antigens. Sustained expansion occurs only when innate immune sensors are activated by microbial stimuli or by adjuvants, which has important implications for vaccination. The molecular identity of the signals controlling sustained T-cell responses is not fully clear. Here, we describe a prominent role for the Notch pathway in this process. Coactivation of Notch allows accumulation of far greater numbers of activated CD4(+) T cells than stimulation via T-cell receptor and classic costimulation alone. Notch does not overtly affect cell cycle entry or progression of CD4(+) T cells. Instead, Notch protects activated CD4(+) T cells against apoptosis after an initial phase of clonal expansion. Notch induces a broad antiapoptotic gene expression program that protects against intrinsic, as well as extrinsic, apoptosis pathways. Both Notch1 and Notch2 receptors and the canonical effector RBPJ (recombination signal binding protein for immunoglobulin kappa J region) are involved in this process. Correspondingly, CD4(+) T-cell responses to immunization with protein antigen are strongly reduced in mice lacking these components of the Notch pathway. Our findings, therefore, show that Notch controls the magnitude of CD4(+) T-cell responses by promoting cellular longevity.

The transcription factor Spi-B regulates human plasmacytoid dendritic cell survival through direct induction of the antiapoptotic gene BCL2-A1.

Plasmacytoid dendritic cells (pDCs) selectively express Toll-like receptor (TLR)-7 and TLR-9, which allow them to rapidly secrete massive amounts of type I interferons after sensing nucleic acids derived from viruses or bacteria. It is not completely understood how development and function of pDCs are controlled at the transcriptional level. One of the main factors driving pDC development is the ETS factor Spi-B, but little is known about its target genes. Here we demonstrate that Spi-B is crucial for the differentiation of hematopoietic progenitor cells into pDCs by controlling survival of pDCs and its progenitors. In search for Spi-B target genes, we identified the antiapoptotic gene Bcl2-A1 as a specific and direct target gene, thereby consolidating the critical role of Spi-B in cell survival.

Fate-Mapping Macrophages: From Ontogeny to Functions.

Macrophages are vital to the physiological function of most tissues, but also contribute to disease through a multitude of pathological roles. They are thus highly plastic and heterogeneous. It is now well recognized that macrophages develop from several distinct progenitors from embryogenesis onwards and extending throughout life. Tissue-resident macrophages largely originate from embryonic sources and in many cases self-maintain independently without monocyte input. However, in certain tissues, monocyte-derived macrophages replace these over time or as a result of tissue injury and inflammation. This additional layer of heterogeneity has introduced many questions regarding the influence of origin on fate and function of macrophages in health and disease. To comprehensively address these questions, appropriate methods of tracing macrophage ontogeny are required. This chapter explores why ontogeny is of vital importance in macrophage biology and how to delineate macrophage populations by origin through genetic fate mapping. First, we summarize the current view of macrophage ontogeny and briefly discuss how origin may influence macrophage function in homeostasis and pathology. We go on to make the case for genetic fate mapping as the gold standard and briefly review different fate-mapping models. We then put forward our recommendations for fate-mapping strategies best suited to answer specific research questions and finally discuss the strengths and limitations of currently available models.

Bcl-2 supports survival and metabolic fitness of quiescent tissue-resident ILC3.

Group 3 innate lymphoid cells (ILC3) are potent effector cells with critical roles in enforcing immunity, barrier integrity and tissue homeostasis along the gastrointestinal tract. ILC3 are considered primarily tissue-resident cells, seeding the gastrointestinal tract during embryonic stages and early life. However, the mechanisms through which mature ILC3 are maintained within adult tissues are poorly understood. Here, we report that lymphoid tissue-inducer-like (LTi-like) ILC3 exhibit minimal turnover in the healthy adult intestinal tract, persist for extended periods of time, and display a quiescent phenotype. Strikingly, during enteric bacterial infection LTi-like ILC3 also exhibit negligible hematopoietic replenishment and remain non-proliferative, despite robustly producing cytokines. Survival of LTi-like ILC3 was found to be dependent upon the balance between the metabolic activity required to drive effector function and anti-apoptotic programs. Notably, the pro-survival protein B-cell lymphoma-2 (Bcl-2) was required for the survival of LTi-like ILC3 ex vivo but was rendered partially dispensable if mitochondrial respiration was inhibited. Together we demonstrate LTi-like ILC3 are a tissue-resident, quiescent population that persist independently of hematopoietic replenishment to survive within the intestinal microenvironment.

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration after radiation-induced injury.

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant immune cell in the salivary glands and are attractive therapeutic targets due to their unrivaled capacity to drive tissue repair. Yet, the nature and role of macrophages in salivary gland homeostasis and how they may contribute to tissue repair after injury are not well understood. Here, we show that at least two phenotypically and transcriptionally distinct CX3CR1+ macrophage populations are present in the adult salivary gland, which occupy anatomically distinct niches. CD11c+CD206-CD163- macrophages typically associate with gland epithelium, whereas CD11c-CD206+CD163+ macrophages associate with blood vessels and nerves. Using a suite of complementary fate mapping systems, we show that there are highly dynamic changes in the ontogeny and composition of salivary gland macrophages with age. Using an in vivo model of radiation-induced salivary gland injury combined with genetic or antibody-mediated depletion of macrophages, we demonstrate an essential role for macrophages in clearance of cells with DNA damage. Furthermore, we show that epithelial-associated macrophages are indispensable for effective tissue repair and gland function after radiation-induced injury, with their depletion resulting in reduced saliva production. Our data, therefore, provide a strong case for exploring the therapeutic potential of manipulating macrophages to promote tissue repair and thus minimize salivary gland dysfunction after radiotherapy.

Landscape of mast cell populations across organs in mice and humans.

Mast cells (MCs) are tissue-resident immune cells that exhibit homeostatic and neuron-associated functions. Here, we combined whole-tissue imaging and single-cell RNA sequencing datasets to generate a pan-organ analysis of MCs in mice and humans at steady state. In mice, we identify two mutually exclusive MC populations, MrgprB2+ connective tissue-type MCs and MrgprB2neg mucosal-type MCs, with specific transcriptomic core signatures. While MrgprB2+ MCs develop in utero independently of the bone marrow, MrgprB2neg MCs develop after birth and are renewed by bone marrow progenitors. In humans, we unbiasedly identify seven MC subsets (MC1-7) distributed across 12 organs with different transcriptomic core signatures. MC1 are preferentially enriched in the bladder, MC2 in the lungs, and MC4, MC6, and MC7 in the skin. Conversely, MC3 and MC5 are shared by most organs but not skin. This comprehensive analysis offers valuable insights into the natural diversity of MC subtypes in both mice and humans.

Developmental programming of macrophages by early life adversity.

Macrophages are central elements of all organs, where they have a multitude of physiological and pathological functions. The first macrophages are produced during fetal development, and most adult organs retain populations of fetal-derived macrophages that self-maintain without major input of hematopoietic stem cell-derived monocytes. Their developmental origins make macrophages highly susceptible to environmental perturbations experienced in early life, in particular the fetal period. It is now well recognized that such adverse developmental conditions contribute to a wide range of diseases later in life. This chapter explores the notion that macrophages are key targets of environmental adversities during development, and mediators of their long-term impact on health and disease. We first briefly summarize our current understanding of macrophage ontogeny and their biology in tissues and consider potential mechanisms by which environmental stressors may mediate fetal programming. We then review evidence for programming of macrophages by adversities ranging from maternal immune activation and diet to environmental pollutants and toxins, which have disease relevance for different organ systems. Throughout this chapter, we contemplate appropriate experimental strategies to study macrophage programming. We conclude by discussing how our current knowledge of macrophage programming could be conceptualized, and finally highlight open questions in the field and approaches to address them.