Klein, F., et al., 2022. Dntt-fate mapping redefines developmental hierarchy and lineage specification of hematopoietic progenitors. Nature Immunology , 23 , pp. 505-17.
Saba, Y., et al., 2022. Early antitumor activity of oral Langerhans cells is compromised by a carcinogen. Proc Nail Acad Sci U S A . , 119 (3) , pp. e2118424119. Publisher's VersionAbstract
Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αβT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.
T, B., et al., 2021. Intradermal lipopolysaccharide challenge as an acute in vivo inflammatory model in healthy volunteers. British Journal of Clinical Pharmacology , Publisher's VersionAbstract

Aims: Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies.

Methods: Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate.

Results: LPS elicited a visible response and returned to baseline at 48 hours. Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1β, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells.

Discussion: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies.

H, S., et al., 2021. Characterization of Human Dendritic Cell Subsets in the Gingiva of Chronic Periodontitis. Journal of Dental Research , 100 (12) , pp. 1330-1336. Publisher's VersionAbstract
As the most potent cells activating and polarizing naive T cells, dendritic cells (DCs) are of major importance in the induction of immunity and tolerance. DCs are a heterogeneous population of antigen-presenting cells that are widely distributed in lymphoid and nonlymphoid tissues. Murine studies have highlighted the important role of oral DCs and Langerhans cells (LCs) in orchestrating the physiological homeostasis of the oral mucosa. DCs are also critically involved in pathological conditions such as periodontal diseases, in which gingival DCs appear to have special localization and function. While the characterization of human DCs in health and disease has been extensively investigated in various tissues, this topic was rarely studied in human gingiva. Here, we employed an up-to-date approach to characterize by flow cytometry the gingival DCs of 27 healthy subjects and 21 periodontal patients. Four distinct subsets of mononuclear phagocytes were identified in healthy gingiva: conventional DC type 1 (cDC1), cDC2, plasmacytoid DCs (pDCs), and LCs. In periodontitis patients, the frequencies of gingival LCs and pDCs were dysregulated, as LCs decreased, whereas pDCs increased in the diseased gingiva. This shift in the prevalence of DCs was accompanied by increased expression of the proinflammatory cytokines interleukin (IL)–1β, interferon (IFN)–α, and IFN-γ, while the anti-inflammatory cytokine IL-10 was suppressed. We further found that smoking, a known risk factor of periodontitis, specifically reduces gingival LCs in healthy individuals, indicating a possible role of LCs in the elevated severity of periodontitis in smokers. Collectively, this work reveals the various DC subsets residing in the human gingiva and the impact of periodontitis, as well as smoking, on the prevalence of each subset. Our findings provide a foundation toward understanding the role of human DCs in orchestrating physiological oral immunity and set the stage for the evaluation and modulation of shifts in immunity associated with periodontitis.
Patel, A.A., Ginhoux, F. & Yona, S., 2021. Monocytes, Macrophages, Dendritic Cells and Neutrophils: an update on lifespan kinetics in health and disease. Immunology.Abstract
Phagocytes form a family of immune cells that play a crucial role in tissue maintenance and help orchestrate the immune response. This family of cells can be separated by their nuclear morphology into mononuclear and polymorphonuclear phagocytes. The generation of these cells in the bone marrow, to the blood and finally into tissues is a tightly regulated process. Ensuring the adequate production of these cells and their timely removal, is key for both the initiation and resolution of inflammation. Insight into the kinetic profiles of innate myeloid cells during steady state and pathology will permit the rational development of therapies to boost the production of these cells in times of need or reduce them when detrimental.
Pallett, L.J., et al., 2020. Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes. Journal of Experimental Medicine. Publisher's VersionAbstract
The human liver contains specialized subsets of mononuclear phagocytes (MNPs) and T cells, but whether these have definitive features of tissue residence (long-term retention, lack of egress) and/or can be replenished from the circulation remains unclear. Here we addressed these questions using HLA-mismatched liver allografts to discriminate the liver-resident (donor) from the infiltrating (recipient) immune composition. Allografts were rapidly infiltrated by recipient leukocytes, which recapitulated the liver myeloid and lymphoid composition, and underwent partial reprogramming with acquisition of CD68/CD206 on MNPs and CD69/CD103 on T cells. The small residual pool of donor cells persisting in allografts for over a decade contained CX3CR1hi/CD163hi/CD206hi Kupffer cells (KCs) and CXCR3hi tissue-resident memory T cells (TRM). CD8+ TRM were found in the local lymph nodes but were not detected egressing into the hepatic vein. Our findings inform organ transplantation and hepatic immunotherapy, revealing remarkably long-lived populations of KCs and TRM in human liver, which can be additionally supplemented by their circulating counterparts.
Giladi, A., et al., 2020. Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation. Nature Immunology , 21 , pp. 535-34. Publisher's VersionAbstract
Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10+ and Saa3+ monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10+ and Saa3+ pathogenic cells were not derived from Ly6C+ monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10+ and Saa3+ monocytes, could be used for targeted therapeutic interventions.
Patel, A.A. & Yona, S., 2019. Inherited and Environmental Factors Influence Human Monocyte Heterogeneity. Front Immunol , 10 , pp. 2581.Abstract
Blood monocytes develop in the bone marrow before being released into the peripheral circulation. The circulating monocyte pool is composed of multiple subsets, each with specialized functions. These cells are recruited to repopulate resident monocyte-derived cells in the periphery and also to sites of injury. Several extrinsic factors influence the function and quantity of monocytes in the blood. Here, we outline the impact of sex, ethnicity, age, sleep, diet, and exercise on monocyte subsets and their function, highlighting that steady state is not a single physiological condition. A clearer understanding of the relationship between these factors and the immune system may allow for improved therapeutic strategies.
Mildner, A. & Yona, S., 2019. Mapping the lung. Science , 363 , pp. 1154-1155.
Janela, B., et al., 2019. A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFalpha-Mediated Recruitment of Neutrophils. Immunity , 50 , pp. 1069-1083 e8.Abstract
Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor alpha (VEGF-alpha) by a minor subset of activated EpCAM(+)CD59(+)Ly-6D(+) cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guerin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.
Gill, U.S., et al., 2019. Fine needle aspirates comprehensively sample intrahepatic immunity. Gut , 68 , pp. 1493-1503.Abstract
OBJECTIVE: In order to refine new therapeutic strategies in the pipeline for HBV cure, evaluation of virological and immunological changes compartmentalised at the site of infection will be required. We therefore investigated if liver fine needle aspirates (FNAs) could comprehensively sample the local immune landscape in parallel with viable hepatocytes. DESIGN: Matched blood, liver biopsy and FNAs from 28 patients with HBV and 15 without viral infection were analysed using 16-colour multiparameter flow cytometry. RESULTS: The proportion of CD4 T, CD8 T, Mucosal Associated Invariant T cell (MAIT), Natural Killer (NK) and B cells identified by FNA correlated with that in liver biopsies from the same donors. Populations of Programmed Death-1 (PD-1)(hi)CD39(hi) tissue-resident memory CD8 T cells (CD69(+)CD103(+)) and liver-resident NK cells (CXCR6(+)T-bet(lo)Eomes(hi)), were identified by both FNA and liver biopsy, and not seen in the blood. Crucially, HBV-specific T cells could be identified by FNAs at similar frequencies to biopsies and enriched compared with blood. FNAs could simultaneously identify populations of myeloid cells and live hepatocytes expressing albumin, Scavenger Receptor class B type 1 (SR-B1), Programmed Death-Ligand 1 (PD-L1), whereas hepatocytes were poorly viable after the processing required for liver biopsies. CONCLUSION: We demonstrate for the first time that FNAs identify a range of intrahepatic immune cells including locally resident sentinel HBV-specific T cells and NK cells, together with PD-L1-expressing hepatocytes. In addition, we provide a scoring tool to estimate the extent to which an individual FNA has reliably sampled intrahepatic populations rather than contaminating blood. The broad profiling achieved by this less invasive, rapid technique makes it suitable for longitudinal monitoring of the liver to optimise new therapies for HBV.
Foote, J.R., et al., 2019. Variations in the Phagosomal Environment of Human Neutrophils and Mononuclear Phagocyte Subsets. Front Immunol , 10 , pp. 188 *Joint Senior Author .Abstract
The phagosome microenvironment maintains enzyme activity and function. Here we compared the phagosomal pH of human neutrophils, monocytes, dendritic cells (DC), and monocyte-derived cells. An unexpected observation was the striking difference in phagosomal environment between the three monocytes subsets. Classical monocytes and neutrophils exhibited alkaline phagosomes, yet non-classical monocytes had more acidic phagosomes, while intermediate monocytes had a phenotype in-between. We next investigated the differences between primary naive DC vs. in vitro monocyte-derived DC (MoDC) and established that both these cells had acidic phagosomal environments. Across all phagocytes, alkalinization was dependent upon the activity of the NADPH oxidase activity, demonstrated by the absence of NADPH oxidase from a patient with chronic granulomatous disease (CGD) or the use of a pharmacological inhibitor, diphenylene iodonium (DPI). Interestingly, MoDC stimulated with bacterial lipopolysaccharide had increased phagosomal pH. Overall, the increase in alkalinity within the phagosome was associated with increased oxidase activity. These data highlight the heterogeneous nature and potential function of phagocytic vacuoles within the family of mononuclear phagocytes.
Yona, S. & Mildner, A., 2018. Good things come in threes. Science Immunology , 3.Abstract
Ectopic expression of PU.1, IRF8, and BATF3 reprograms mouse and human fibroblasts into dendritic cells. See related Research Article by Rosa et al.
Stremmel, C., et al., 2018. Yolk sac macrophage progenitors traffic to the embryo during defined stages of development. Nat Commun , 9 , pp. 75.Abstract
Tissue macrophages in many adult organs originate from yolk sac (YS) progenitors, which invade the developing embryo and persist by means of local self-renewal. However, the route and characteristics of YS macrophage trafficking during embryogenesis are incompletely understood. Here we show the early migration dynamics of YS-derived macrophage progenitors in vivo using fate mapping and intravital microscopy. From embryonic day 8.5 (E8.5) CX3CR1+ pre-macrophages are present in the mouse YS where they rapidly proliferate and gain access to the bloodstream to migrate towards the embryo. Trafficking of pre-macrophages and their progenitors from the YS to tissues peaks around E10.5, dramatically decreases towards E12.5 and is no longer evident from E14.5 onwards. Thus, YS progenitors use the vascular system during a restricted time window of embryogenesis to invade the growing fetus. These findings close an important gap in our understanding of the development of the innate immune system.
Maini, A., et al., 2018. Monocyte and Neutrophil Isolation, Migration, and Phagocytosis Assays. Curr Protoc Immunol , pp. e53.Abstract
This article describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell phagocytosis, migration, and polarization. The method employed here for the isolation of naive phagocytes overcomes many of the difficulties previously encountered concerning phagocyte activation. Three in vitro protocols are provided for the analysis of cell migration, one requiring no specialized equipment, one requiring a modified Boyden chamber, and the other employing a flow chamber, which measures cell adhesion, rolling, and migration. Three in vitro protocols to examine phagocytosis have been included in this updated version. Finally, a method is provided for imaging polarized cells by confocal microscopy. (c) 2018 by John Wiley & Sons, Inc.
Guilliams, M., Mildner, A. & Yona, S., 2018. Developmental and Functional Heterogeneity of Monocytes. Immunity , 49 , pp. 595-613.Abstract
Novel experimental approaches such as fate-mapping and single-cell analysis have brought fresh insight into monocyte development and function over the past decade and helped redefine the monocyte field. Monocytes are now known to consist of multiple subsets generated through distinct developmental pathways with diverse functional specializations. Their fates under homeostatic conditions include the accumulation in peripheral reservoirs and the engraftment into certain resident macrophage pools. Under pathological conditions, monocytes acquire inflammatory effector functions, but can also develop regulatory properties essential for tissue repair. Importantly, monocytes recruited during inflammation are often functionally distinct from resident macrophages or conventional dendritic cells. Here we outline emerging concepts in monocyte heterogeneity, emergency monopoiesis, and trained immunity and discuss how these bring new perspectives to monocyte research.
Haimon, Z., et al., 2018. Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies. Nat Immunol , 19 , pp. 636-644.Abstract
Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.
Wolf, Y., et al., 2017. Autonomous TNF is critical for in vivo monocyte survival in steady state and inflammation. J Exp Med , 214 , pp. 905-917.Abstract
Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6C(hi) effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.
Varol, D., et al., 2017. Dicer Deficiency Differentially Impacts Microglia of the Developing and Adult Brain. Immunity , 46 , pp. 1030-1044 e8.Abstract
Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.
Patel, A.A., et al., 2017. The fate and lifespan of human monocyte subsets in steady state and systemic inflammation. J Exp Med , 214 , pp. 1913-1923.Abstract
In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans ( approximately 4 and approximately 7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.