Niche signals and transcription factors involved in tissue-resident macrophage development

Tissue-resident macrophages form an essential part of the first line of defense in all tissues of the body. Next to their immunological role, they play an important role in maintaining tissue homeostasis. Recently, it was shown that they are primarily of embryonic origin. During embryogenesis, precursors originating in the yolk sac and fetal liver colonize the embryonal tissues where they develop into mature tissue-resident macrophages. Their development is governed by two distinct sets of transcription factors. First, in the pre-macrophage stage, a core macrophage program is established by lineage-determining transcription factors. Under the influence of tissue-specific signals, this core program is refined by signal-dependent transcription factors. This nurturing by the niche allows the macrophages to perform tissue-specific functions. In the last 15 years, some of these niche signals and transcription factors have been identified. However, detailed insight in the exact mechanism of development is still lacking

The Peyer's patch mononuclear phagocyte system at steady state and during infection

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer's patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle- associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed

Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD

Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-alpha (LXR-alpha). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-alpha via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity

Phenotypical, ontogeny and functional characterization of the Peyer's patch mononuclear phagocyte system

Les plaques de Peyer (PP) sont les principaux sites inducteurs de la réponse immunitaire mucosale.L’épithélium associé aux follicules comprend des cellules épithéliales spécifiques, appelées cellules M et spécialisées dans le transport du matériel présent dans la lumière intestinale vers le dôme sous épithélial (SED) où il sera pris en charge par les cellules du système phagocytaire mononuclée (MPS) qui orchestreront ensuite les réponses immunitaires mucosales.Nous avons effectué une analyse complète du phénotype, de la distribution, de l’ontogénie, de la fonction et des profils transcriptomiques du MPS des PP. Nous avons montré que les monocytes donnent naissance à deux populations: les lysoDC et les lysoMac. La première exprime de fort niveau de CMH-II et de molécules de costimulation, a une courte durée de vie et est capable d’activer les lymphocytes T naïfs pour sécréter de l’IFNγ tandis que la deuxième exprime faiblement le CMH-II, à une longue durée de vie et n’est pas capable d’activer les LT naïfs. Ces deux populations ont toutefois des propriétés communes de phagocytose et de défense innée contre les virus et les bactéries entéropathogènes. Nous avons identifié deux populations distinctes de lysoMac selon l’expression de Tim4: les lysoMac Tim4+ situés dans l’IFR et la partie inférieure du follicule ; les lysoMac Tim4- situés dans le SED et la partie supérieure du follicule. Nous avons aussi déterminé 4 états de maturation pour les lysoDC suivant l’expression d’Emb, Jam-A et CD24. Nous avons également redéfini la localisation de chaque population du MPS des PP fournissant ainsi une base solide pour étudier le rôle de chacun de ses membres dans l’immunité mucosale.Peyer’s patches (PPs) are primary inductive sites of mucosal immunity. The follicle-associated epithelium contains specialized epithelial cells, called M cells, that bind and rapidly transport microorganisms from the lumen to the subepithelial dome (SED) where they are internalized by cells of the mononuclear phagocyte system (MPS) which are involved in the initiation of the mucosal immune responses. MPS comprise monocytes, macrophages (Mφ) and dendritic cells (DC). Here, we provide a comprehensive analysis of the phenotype, distribution, ontogeny, function, and transcriptional profile of PP MPS. We show that monocyte give rise to two different cell populations named lysoDC and lysoMac. The former express high levels of MHCII and costimulatory molecules, have a short half life and are able to prime naïve T cells for IFNγ production while the latter display low levels of MHCII, have a long half life and are unable to prime naïve T cells efficiently. However, these two cell populations share common features such as phagocytosis and antimicrobial defense mechanisms. LysoMac can be separated in two subpopulations according to Tim4 expression: Tim4+ lysoMac located in the IFR and the lower part of the follicle; Tim4- lysoMac located in the SED and upper part of the follicle. LysoDC can be separated in four different maturation stages according to Emb, Jam-A and CD24 expression. Finally, we redefined the location of each PP MPS population. In summary, we provide a comprehensive map of the PP MPS which will allow to study its role in mucosal immune response initiation and regulation

Developmental control of macrophage function

The combination between novel fate-mapping tools and single cell RNA-sequencing technology has revealed the presence of multiple macrophage progenitors. This raises the fascinating possibility that what was once perceived as immense functional plasticity of macrophages could in fact come down to separate macrophage subsets performing distinct functions because of their differential cellular origin. The question of macrophage plasticity versus macrophage heterogeneity is broader than the difference between macrophages of embryonic or adult hematopoietic origin and is particularly relevant in the context of inflammation. In this manuscript, we review the potential impact of cellular origin on the function of macrophages. We also highlight the need for novel 'functional fate-mapping' tools that would reveal the history of the functional state of macrophages, rather than their cellular origin, in order to finally study their true plasticity in vivo

Establishment and maintenance of the macrophage niche

Self-maintaining resident macrophages populate all mammalian organs. In addition to their role as immune sentinels, macrophages perform day-to-day functions essential to tissue homeostasis. The homeostatic functions of macrophages are regulated by so-called tissular "niches'' that control the size of the macrophage population and imprint their tissue-specific identity. Here, we review the mechanisms underlying self-maintenance of distinct macrophage populations and outline the organizing principles of the macrophage niche. We examine recent studies that uncovered mutually beneficial cell-cell circuits established between macrophages and their niche and propose a modular view of tissues that integrates the resident macrophage as an essential component of each individual module. Manipulating macrophage niche cells to control the function of resident macrophages in vivo might have therapeutic value in various disease settings

Some news from the unknown soldier, the Peyer's patch macrophage

In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintenance of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer's patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30 years ago, MF of PP escaped any kind of phenotypic description and remained "unknown" for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions

Gene expression profiling of the Peyer's patch mononuclear phagocyte system

Peyer's patches (PPs) are primary inductive sites of mucosal immunity. The PP mononuclear phagocyte system, which encompasses both dendritic cells (DCs) and macrophages, is essential for the initiation of the mucosal immune response. We recently developed a method to isolate each mononuclear phagocyte subset of PP (Bonnardel et al., 2015). We performed a transcriptional analysis of three of these subsets: the CD11b+ conventional DC, the lysozyme-expressing monocyte-derived DC termed LysoDC and the CD11chi lysozyme-expressing macrophages. Here, we provide details of the gating strategy we used to isolate each phagocyte subset and show the quality controls and analysis associated with our gene array data deposited into Gene Expression Omnibus (GEO) under GSE65514

A workflow for 3D-CLEM investigating liver tissue

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. Lay Description As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10(-9) m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail