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Unlike sessile macrophages that occupy specialized tissue niches, non-classical monocytes (NCMs)-circulating phagocytes that patrol and cleanse the luminal surface of the vascular tree-are characterized by constant movement. Here, we examined the nature of the NCM's nurturing niche. Expression of the growth factor CSF1 on endothelial cells was required for survival of NCMs in the bloodstream. Lack of endothelial-derived CSF1 did not affect blood CSF1 concentration, suggesting that NCMs rely on scavenging CSF1 present on endothelial cells. Deletion of the transmembrane chemokine and adhesion factor CX3CL1 on endothelial cells impaired NCM survival. Mechanistically, endothelial-derived CX3CL1 and integrin subunit alpha L (ITGAL) facilitated the uptake of CSF1 by NCMs. CSF1 was produced by all tissular endothelial cells, and deletion of Csf1 in all endothelial cells except bone marrow sinusoids impaired NCM survival, arguing for a model where the full vascular tree acts as a niche for NCMs and where survival and patrolling function are connected., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Testis-resident macrophages are first responders of the innate immune system against pathogens. They also exert day-to-day functions that are poorly understood. To study testis macrophages, several techniques are used, among which we can find flow cytometry.Flow cytometry is a powerful tool that enables analysis of macrophages at a cellular as well as population level. To analyze testis macrophages using flow cytometry, a specific tissue processing is necessary to extract them. In this protocol, we explain how to extract and analyze the distinct macrophage populations., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2023 Tauber et al.)

Tingible body macrophages in lymph node are involved in cleaning up debris from apoptotic B cells. Gurwisz et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20222173) and Grootveld et al. (2023. Cell.https://doi.org/10.1016/j.cell.2023.02.004) report how tingible body macrophages, originating from tissue-resident macrophages, clear apoptotic B cells in the germinal center using a "stand-hunting" strategy., (© 2023 Baudon and Bajenoff.)

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206 lo ESAM - population (KC1) and a minor CD206 hi ESAM + population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions., Competing Interests: Declaration of interests C.B., M.A., and F.G. are inventors on a patent filed, owned, and managed by A(∗)ccelerate technologies Pte Ltd, A-STAR, Singapore, on technology related to the work presented in this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8 + T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8 + T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity., Competing Interests: Declaration of interests M.I. participates in advisory boards and consultancies for Gilead Sciences, Roche, Third Rock Ventures, Amgen, and Allovir. L.G.G is a member of the board of directors at Genenta Science and Epsilon Bio and participates in advisory boards and consultancies for Gilead Sciences, Roche, and Arbutus Biopharma. M.I. and L.G.G. are inventors on patents filed, owned, and managed by San Raffaele Scientific Institute, Vita-Salute San Raffaele University, and the Telethon Foundation on technology related to work discussed in this manuscript (WO2020/016434, WO2020/016427, WO2020/030781, WO2020/234483, European Union [EU] patent applications 19211249.8 and 20156716.1, and U.K. patent application 1907493.9). F.G. is a member of the Immunity advisory board., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Resident macrophages are abundant in the bladder, playing key roles in immunity to uropathogens. Yet, whether they are heterogeneous, where they come from, and how they respond to infection remain largely unknown. We identified two macrophage subsets in mouse bladders, MacM in muscle and MacL in the lamina propria, each with distinct protein expression and transcriptomes. Using a urinary tract infection model, we validated our transcriptomic analyses, finding that MacM macrophages phagocytosed more bacteria and polarized to an anti-inflammatory profile, whereas MacL macrophages died rapidly during infection. During resolution, monocyte-derived cells contributed to tissue-resident macrophage pools and both subsets acquired transcriptional profiles distinct from naïve macrophages. Macrophage depletion resulted in the induction of a type 1-biased immune response to a second urinary tract infection, improving bacterial clearance. Our study uncovers the biology of resident macrophages and their responses to an exceedingly common infection in a largely overlooked organ, the bladder., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence., Competing Interests: Disclosures: Dr. Etzerodt reported personal fees from Stipe Therapeutics outside the submitted work. No other disclosures were reported., (© 2020 Etzerodt et al.)

Self-maintaining resident macrophages populate all mammalian organs. In addition to their role as immune sentinels, macrophages also 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 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1 -/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7 + stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1 lo MHCII hi CX3CR1 hi (Lyve1 lo MHCII hi ) and Lyve1 hi MHCII lo CX3CR1 lo (Lyve1 hi MHCII lo ) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion ( Slco2b1 flox/DTR ), we found that the absence of Lyve1 hi MHCII lo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169 + MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

BACKGROUND Migration of leukocytes into airways is the hallmark of allergic asthma. The aim of this study was to target the pathological process using pantethine, a pleiotropic natural compound which has been recently shown to down-regulate chemokine-driven T cell migration. MATERIAL AND METHODS Mice were sensitized to the Leishmania LACK antigen, then treated or not treated with pantethine and exposed to LACK or saline aerosol. After sacrifice of the animals, cells in the bronchoalveolar lavage were analyzed and inflammatory parameters were determined to evaluate inflammation seriousness. RESULTS As compared to untreated animals, pantethine-treated animals displayed a moderated response to the allergen, as documented by decreased infiltration of inflammatory cells (all types), in addition to reduced levels of lung Th2 cytokines and circulating LACK-specific IgE. CONCLUSIONS These data reveal the potential therapeutic importance of pantethine to moderate allergic asthma pathology. The compound has been previously shown to exert a broad range of protective activity in animals and in humans, with few or no adverse effects.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Psoriasis is a chronic inflammatory skin disease of unknown etiology. Previous studies showed that short-term, 5-7 d-long application of imiquimod (IMQ), a TLR7 agonist, to the skin of mice triggers a psoriasis-like inflammation. In the current study, by applying IMQ for 14 consecutive d, we established an improved mouse psoriasis-like model in that it recapitulated many of the clinical and cellular hallmarks observed in human patients during both the early-onset and the late-stable phase of psoriasis. Although macrophages and dendritic cells (DCs) have been proposed to drive the psoriatic cascade, their largely overlapping phenotype hampered studying their respective role. Based on our ability to discriminate Langerhans cells (LCs), conventional DCs, monocytes, monocyte-derived DCs, macrophages, and plasmacytoid DCs in the skin, we addressed their dynamics during both phases of our biphasic psoriasis-like model. Plasmacytoid DCs were not detectable during the whole course of IMQ treatment. During the early phase, neutrophils infiltrated the epidermis, whereas monocytes and monocyte-derived DCs were predominant in the dermis. During the late phase, LCs and macrophage numbers transiently increased in the epidermis and dermis, respectively. LC expansion resulted from local proliferation, a conclusion supported by global transcriptional analysis. Genetic depletion of LCs permitted to evaluate their function during both phases of the biphasic psoriasis-like model and demonstrated that their absence resulted in a late phase that is associated with enhanced neutrophil infiltration. Therefore, our data support an anti-inflammatory role of LCs during the course of psoriasis-like inflammation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation., (© 2014 Molawi et al.)

Dendritic cells (DC) are required for priming antigen-specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto-reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti-inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen-specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.

The development and maintenance of secondary lymphoid organs, such as lymph nodes, occur in a highly coordinated manner involving lymphoid chemokine production by stromal cells. Although developmental pathways inducing lymphoid chemokine production during organogenesis are known, signals maintaining cytokine production in adults are still elusive. In this study, we show that thrombomodulin and platelet-derived growth factor receptor α identify a population of fibroblastic reticular cells in which chemokine secretion is controlled by JAM-C. We demonstrate that Jam-C-deficient mice and mice treated with Ab against JAM-C present significant decreases in stromal cell-derived factor 1α (CXCL12), CCL21, and CCL19 intranodal content. This effect is correlated with reduced naive T cell egress from lymph nodes of anti-JAM-C-treated mice.

After entry into thymus, T cell progenitors migrate in the cortex and the medulla while completing their education. Recent reports have documented the dynamic and tortuous behavior of thymocytes. However, other than chemokines and/or segregated thymic substrates, the factors contributing to the dynamic patterns of thymocyte movement are poorly characterized. By combining confocal and dynamic two-photon microscopy, we demonstrate that thymocytes continuously migrate on thymic stromal cell networks. In addition to constituting "roads" for thymocytes, we observed that these networks also provide a scaffold on which dendritic cells attach themselves. These results highlight the central role of stromal microanatomy in orchestrating the multiple cellular interactions necessary for T cell migration/development within the thymus.