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.

The Ins and Outs of Central Nervous System Inflammation-Lessons Learned from Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease that is characterized by the inappropriate invasion of lymphocytes and monocytes into the central nervous system (CNS), where they orchestrate the demyelination of axons, leading to physical and cognitive disability. There are many reasons immunologists should be interested in MS. Aside from the fact that there is still significant unmet need for patients living with the progressive form of the disease, MS is a case study for how immune cells cross CNS barriers and subsequently interact with specialized tissue parenchymal cells. In this review, we describe the types of immune cells that infiltrate the CNS and then describe interactions between immune cells and glial cells in different types of lesions. Lastly, we provide evidence for CNS-compartmentalized immune cells and speculate on how this impacts disease progression for MS patients.

Hypoxia and Innate Immunity: Keeping Up with the HIFsters.

Recent years have witnessed an emergence of interest in understanding metabolic changes associated with immune responses, termed immunometabolism. As oxygen is central to all aerobic metabolism, hypoxia is now recognized to contribute fundamentally to inflammatory and immune responses. Studies from a number of groups have implicated a prominent role for oxygen metabolism and hypoxia in innate immunity of healthy tissue (physiologic hypoxia) and during active inflammation (inflammatory hypoxia). This inflammatory hypoxia emanates from a combination of recruited inflammatory cells (e.g., neutrophils, eosinophils, and monocytes), high rates of oxidative metabolism, and the activation of multiple oxygen-consuming enzymes during inflammation. These localized shifts toward hypoxia have identified a prominent role for the transcription factor hypoxia-inducible factor (HIF) in the regulation of innate immunity. Such studies have provided new and enlightening insight into our basic understanding of immune mechanisms, and extensions of these findings have identified potential therapeutic targets. In this review, we summarize recent literature around the topic of innate immunity and mucosal hypoxia with a focus on transcriptional responses mediated by HIF.

Nonclassical Monocytes in Health and Disease.

Monocytes are innate blood cells that maintain vascular homeostasis and are early responders to pathogens in acute infections. There are three well-characterized classes of monocytes: classical (CD14+CD16- in humans and Ly6Chi in mice), intermediate (CD14+CD16+ in humans and Ly6C+Treml4+ in mice), and nonclassical (CD14-CD16+ in humans and Ly6Clo in mice). Classical monocytes are critical for the initial inflammatory response. Classical monocytes can differentiate into macrophages in tissue and can contribute to chronic disease. Nonclassical monocytes have been widely viewed as anti-inflammatory, as they maintain vascular homeostasis. They are a first line of defense in recognition and clearance of pathogens. However, their roles in chronic disease are less clear. They have been shown to be protective as well as positively associated with disease burden. This review focuses on the state of the monocyte biology field and the functions of monocytes, particularly nonclassical monocytes, in health and disease.

Antigen-Presenting Cells in the Skin.

Professional antigen-presenting cells (APCs) in the skin include dendritic cells, monocytes, and macrophages. They are highly dynamic, with the capacity to enter skin from the peripheral circulation, patrol within tissue, and migrate through lymphatics to draining lymph nodes. Skin APCs are endowed with antigen-sensing, -processing, and -presenting machinery and play key roles in initiating, modulating, and resolving cutaneous inflammation. Skin APCs are a highly heterogeneous population with functionally specialized subsets that are developmentally imprinted and modulated by local tissue microenvironmental and inflammatory cues. This review explores recent advances that have allowed for a more accurate taxonomy of APC subsets found in both mouse and human skin. It also examines the functional specificity of individual APC subsets and their collaboration with other immune cell types that together promote adaptive T cell and regional cutaneous immune responses during homeostasis, inflammation, and disease.

Innate immune memory after brain injury drives inflammatory cardiac dysfunction.

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

The life-saving benefit of dexamethasone in severe COVID-19 is linked to a reversal of monocyte dysregulation.

Dexamethasone is a life-saving treatment for severe COVID-19, yet its mechanism of action is unknown, and many patients deteriorate or die despite timely treatment initiation. Here, we identify dexamethasone treatment-induced cellular and molecular changes associated with improved survival in COVID-19 patients. We observed a reversal of transcriptional hallmark signatures in monocytes associated with severe COVID-19 and the induction of a monocyte substate characterized by the expression of glucocorticoid-response genes. These molecular responses to dexamethasone were detected in circulating and pulmonary monocytes, and they were directly linked to survival. Monocyte single-cell RNA sequencing (scRNA-seq)-derived signatures were enriched in whole blood transcriptomes of patients with fatal outcome in two independent cohorts, highlighting the potential for identifying non-responders refractory to dexamethasone. Our findings link the effects of dexamethasone to specific immunomodulation and reversal of monocyte dysregulation, and they highlight the potential of single-cell omics for monitoring in vivo target engagement of immunomodulatory drugs and for patient stratification for precision medicine approaches.

The choroid plexus synergizes with immune cells during neuroinflammation.

The choroid plexus (ChP) is a vital brain barrier and source of cerebrospinal fluid (CSF). Here, we use longitudinal two-photon imaging in awake mice and single-cell transcriptomics to elucidate the mechanisms of ChP regulation of brain inflammation. We used intracerebroventricular injections of lipopolysaccharides (LPS) to model meningitis in mice and observed that neutrophils and monocytes accumulated in the ChP stroma and surged across the epithelial barrier into the CSF. Bi-directional recruitment of monocytes from the periphery and, unexpectedly, macrophages from the CSF to the ChP helped eliminate neutrophils and repair the barrier. Transcriptomic analyses detailed the molecular steps accompanying this process and revealed that ChP epithelial cells transiently specialize to nurture immune cells, coordinating their recruitment, survival, and differentiation as well as regulation of the tight junctions that control the permeability of the ChP brain barrier. Collectively, we provide a mechanistic understanding and a comprehensive roadmap of neuroinflammation at the ChP brain barrier.

FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice.

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary ß-glucan-induced inflammatory adaptation.

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages' functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal ß-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, ß-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.

Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving peripheral leukocytes and brain resident cells that contribute to both tissue injury and repair. However, their dynamics and diversity remain poorly understood. To address these limitations, we performed a single-cell transcriptomic study of brain and blood cells 2 or 14 days after ischemic stroke in mice. We observed a strong divergence of post-ischemic microglia, monocyte-derived macrophages and neutrophils over time, while endothelial cells and brain-associated macrophages showed altered transcriptomic signatures at 2 days poststroke. Trajectory inference predicted the in situ trans-differentiation of macrophages from blood monocytes into day 2 and day 14 phenotypes, while neutrophils were projected to be continuously de novo recruited from the blood. Brain single-cell transcriptomes from both female and male aged mice were similar to that of young male mice, but aged and young brains differed in their immune cell composition. Although blood leukocyte analysis also revealed altered transcriptomes after stroke, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification occurs within the brain in the early and recovery phases of ischemic stroke. A portal ( https://anratherlab.shinyapps.io/strokevis/ ) is provided to allow user-friendly access to our data.

Profibrotic monocyte-derived alveolar macrophages are expanded in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19.

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

Vaccination reduces central nervous system IL-1ß and memory deficits after COVID-19 in mice.

Up to 25% of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit postacute cognitive sequelae. Although millions of cases of coronavirus disease 2019 (COVID-19)-mediated memory dysfunction are accumulating worldwide, the underlying mechanisms and how vaccination lowers risk are unknown. Interleukin-1 (IL-1), a key component of innate immune defense against SARS-CoV-2 infection, is elevated in the hippocampi of individuals with COVID-19. Here we show that intranasal infection of C57BL/6J mice with SARS-CoV-2 Beta variant leads to central nervous system infiltration of Ly6Chi monocytes and microglial activation. Accordingly, SARS-CoV-2, but not H1N1 influenza virus, increases levels of brain IL-1ß and induces persistent IL-1R1-mediated loss of hippocampal neurogenesis, which promotes postacute cognitive deficits. Vaccination with a low dose of adenoviral-vectored spike protein prevents hippocampal production of IL-1ß during breakthrough SARS-CoV-2 infection, loss of neurogenesis and subsequent memory deficits. Our study identifies IL-1ß as one potential mechanism driving SARS-CoV-2-induced cognitive impairment in a new mouse model that is prevented by vaccination.

Microbiota regulates intratumoral monocytes to promote anti-tumor immune responses.

The gut microbiota has been shown to promote the efficacy of cancer therapy through regulating adaptive immune responses. In this issue of Cell, Lam et al. provide new evidence demonstrating that specific gut bacteria also reprogram the innate immune tumor microenvironment to enhance the efficacy of cancer therapies.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells.

Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties across different tumors remain elusive. Here, by performing a pan-cancer analysis of single myeloid cells from 210 patients across 15 human cancer types, we identified distinct features of TIMs across cancer types. Mast cells in nasopharyngeal cancer were found to be associated with better prognosis and exhibited an anti-tumor phenotype with a high ratio of TNF+/VEGFA+ cells. Systematic comparison between cDC1- and cDC2-derived LAMP3+ cDCs revealed their differences in transcription factors and external stimulus. Additionally, pro-angiogenic tumor-associated macrophages (TAMs) were characterized with diverse markers across different cancer types, and the composition of TIMs appeared to be associated with certain features of somatic mutations and gene expressions. Our results provide a systematic view of the highly heterogeneous TIMs and suggest future avenues for rational, targeted immunotherapies.