Timing and location dictate monocyte fate and their transition to tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes, and intratumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC.

Deciphering spatial domains from spatial multi-omics with SpatialGlue.

Advances in spatial omics technologies now allow multiple types of data to be acquired from the same tissue slice. To realize the full potential of such data, we need spatially informed methods for data integration. Here, we introduce SpatialGlue, a graph neural network model with a dual-attention mechanism that deciphers spatial domains by intra-omics integration of spatial location and omics measurement followed by cross-omics integration. We demonstrated SpatialGlue on data acquired from different tissue types using different technologies, including spatial epigenome-transcriptome and transcriptome-proteome modalities. Compared to other methods, SpatialGlue captured more anatomical details and more accurately resolved spatial domains such as the cortex layers of the brain. Our method also identified cell types like spleen macrophage subsets located at three different zones that were not available in the original data annotations. SpatialGlue scales well with data size and can be used to integrate three modalities. Our spatial multi-omics analysis tool combines the information from complementary omics modalities to obtain a holistic view of cellular and tissue properties.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

Cebp1 and Cebpß transcriptional axis controls eosinophilopoiesis in zebrafish.

Eosinophils are a group of granulocytes well known for their capacity to protect the host from parasites and regulate immune function. Diverse biological roles for eosinophils have been increasingly identified, but the developmental pattern and regulation of the eosinophil lineage remain largely unknown. Herein, we utilize the zebrafish model to analyze eosinophilic cell differentiation, distribution, and regulation. By identifying eslec as an eosinophil lineage-specific marker, we establish a Tg(eslec:eGFP) reporter line, which specifically labeled cells of the eosinophil lineage from early life through adulthood. Spatial-temporal analysis of eslec+ cells demonstrates their organ distribution from larval stage to adulthood. By single-cell RNA-Seq analysis, we decipher the eosinophil lineage cells from lineage-committed progenitors to mature eosinophils. Through further genetic analysis, we demonstrate the role of Cebp1 in balancing neutrophil and eosinophil lineages, and a Cebp1-Cebpß transcriptional axis that regulates the commitment and differentiation of the eosinophil lineage. Cross-species functional comparisons reveals that zebrafish Cebp1 is the functional orthologue of human C/EBPεP27 in suppressing eosinophilopoiesis. Our study characterizes eosinophil development in multiple dimensions including spatial-temporal patterns, expression profiles, and genetic regulators, providing for a better understanding of eosinophilopoiesis.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

Origin and Heterogeneity of Tissue Myeloid Cells: A Focus on GMP-Derived Monocytes and Neutrophils.

Myeloid cells are a significant proportion of leukocytes within tissues, comprising granulocytes, monocytes, dendritic cells, and macrophages. With the identification of various myeloid cells that perform separate but complementary functions during homeostasis and disease, our understanding of tissue myeloid cells has evolved significantly. Exciting findings from transcriptomics profiling and fate-mapping mouse models have facilitated the identification of their developmental origins, maturation, and tissue-specific specializations. This review highlights the current understanding of tissue myeloid cells and the contributing factors of functional heterogeneity to better comprehend the complex and dynamic immune interactions within the healthy or inflamed tissue. Specifically, we discuss the new understanding of the contributions of granulocyte-monocyte progenitor-derived phagocytes to tissue myeloid cell heterogeneity as well as the impact of niche-specific factors on monocyte and neutrophil phenotype and function. Lastly, we explore the developing paradigm of myeloid cell heterogeneity during inflammation and disease.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Shortened Telomere Length in Sputum Cells of Bronchiectasis Patients is Associated with Dysfunctional Inflammatory Pathways.

Telomere attrition is an established ageing biomarker and shorter peripheral blood leukocyte telomere length has been associated with increased risks of respiratory diseases. However, whether telomere length in disease-relevant sputum immune cells of chronic respiratory disease patients is shortened and which pathways are dysfunctional are not clear. Here we measured telomere length from sputum samples of bronchiectasis and asthmatic subjects and determined that telomere length in sputum of bronchiectasis subjects was significantly shorter (Beta = - 1.167, PAdj = 2.75 × 10-4). We further performed global gene expression analysis and identified genes involved in processes such as NLRP3 inflammasome activation and regulation of adaptive immune cells when bronchiectasis sputum telomere length was shortened. Our study provides insights on dysfunctions related to shortened telomere length in sputum immune cells of bronchiectasis patients.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

Neutrophil subsets and their differential roles in viral respiratory diseases.

Neutrophils play significant roles in immune homeostasis and as neutralizers of microbial infections. Recent evidence further suggests heterogeneity of neutrophil developmental and activation states that exert specialized effector functions during inflammatory disease conditions. Neutrophils can play multiple roles during viral infections, secreting inflammatory mediators and cytokines that contribute significantly to host defense and pathogenicity. However, their roles in viral immunity are not well understood. In this review, we present an overview of neutrophil heterogeneity and its impact on the course and severity of viral respiratory infectious diseases. We focus on the evidence demonstrating the crucial roles neutrophils play in the immune response toward respiratory infections, using influenza as a model. We further extend the understanding of neutrophil function with the studies pertaining to COVID-19 disease and its neutrophil-associated pathologies. Finally, we discuss the relevance of these results for future therapeutic options through targeting and regulating neutrophil-specific responses.

Intrafemoral Delivery of Hematopoietic Progenitors.

Hematopoiesis is a central process and is essential for the replenishment of short-lived leukocytes such as neutrophils. However, the molecular events underlining the developmental transition of quiescent hematopoietic stem cells into downstream progenitors and mature blood cells are not completely understood. Here, we describe the intrafemoral delivery of hematopoietic progenitors as a method to trace their development and differentiation lineage patterns within the bone marrow (BM) niche. Unlike other approaches, the direct adoptive transfer of progenitors into the BM cavity does not require prior irradiation preconditioning of recipient mice, and enables the delivery of lower cell numbers into the marrow space in a minimally perturbed environment. As a demonstrative example, we provide a protocol for the isolation of granulocyte-monocyte progenitors (GMP) by cell sorting, the delivery of these cells into recipient animals by intrafemoral transfer, and finally, the analysis of GMP-derived progenies by flow cytometry.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker for severe COVID-19.

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Fate Mapping via Ms4a3-Expression History Traces Monocyte-Derived Cells.

Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes, and their functions compared to embryonic RTMs remain unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation, and disease are highly debated. Here, we identified Ms4a3 as a specific gene expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3TdT reporter, Ms4a3Cre, and Ms4a3CreERT2 fate-mapping models. These models traced efficiently monocytes and granulocytes, but no lymphocytes or tissue dendritic cells. Using these models, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

Dimensionality reduction for visualizing single-cell data using UMAP.

Advances in single-cell technologies have enabled high-resolution dissection of tissue composition. Several tools for dimensionality reduction are available to analyze the large number of parameters generated in single-cell studies. Recently, a nonlinear dimensionality-reduction technique, uniform manifold approximation and projection (UMAP), was developed for the analysis of any type of high-dimensional data. Here we apply it to biological data, using three well-characterized mass cytometry and single-cell RNA sequencing datasets. Comparing the performance of UMAP with five other tools, we find that UMAP provides the fastest run times, highest reproducibility and the most meaningful organization of cell clusters. The work highlights the use of UMAP for improved visualization and interpretation of single-cell data.

Streamlining volumetric multi-channel image cytometry using hue-saturation-brightness-based surface creation.

Image cytometry is the process of converting image data to flow cytometry-style plots, and it usually requires computer-aided surface creation to extract out statistics for cells or structures. One way of dealing with structures stained with multiple markers in three-dimensional images, is carrying out multiple rounds of channel co-localization and image masking before surface creation, which is cumbersome and laborious. We propose the application of the hue-saturation-brightness color space to streamline this process, which produces complete surfaces, and allows the user to have a global view of the data before flexibly defining cell subsets. Spectral compensation can also be performed after surface creation to accurately resolve different signals. We demonstrate the utility of this workflow in static and dynamic imaging datasets of a needlestick injury on the mouse ear, and we believe this scalable and intuitive approach will improve the ease of performing histocytometry on biological samples.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Dual modal ultra-bright nanodots with aggregation-induced emission and gadolinium-chelation for vascular integrity and leakage detection.

The study of blood brain barrier (BBB) functions is important for neurological disorder research. However, the lack of suitable tools and methods has hampered the progress of this field. Herein, we present a hybrid nanodot strategy, termed AIE-Gd dots, comprising of a fluorogen with aggregation-induced emission (AIE) characteristics as the core to provide bright and stable fluorescence for optical imaging, and gadolinium (Gd) for accurate quantification of vascular leakage via inductively-coupled plasma mass spectrometry (ICP-MS). In this report, we demonstrate that AIE-Gd dots enable direct visualization of brain vascular networks under resting condition, and that they form localized punctate aggregates and accumulate in the brain tissue during experimental cerebral malaria, indicative of hemorrhage and BBB malfunction. With its superior detection sensitivity and multimodality, we hereby propose that AIE-Gd dots can serve as a better alternative to Evans blue for visualization and quantification of changes in brain barrier functions.