The Myeloid Cell Compartment-Cell by Cell.

Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of cellular ontogeny, activation, differentiation, and tissue-specific functions of myeloid cells have been revisited during the last years with surprising results; for example, most tissue macrophages are yolk sac derived, monocytes and macrophages follow a multidimensional model of activation, and tissue signals have a significant impact on the functionality of all these cells. While these exciting results have brought these cells back to center stage, their enormous plasticity and heterogeneity, during both homeostasis and disease, are far from understood. At the same time, the ongoing revolution in single-cell genomics, with single-cell RNA sequencing (scRNA-seq) leading the way, promises to change this. Prevailing models of hematopoiesis with distinct intermediates are challenged by scRNA-seq data suggesting more continuous developmental trajectories in the myeloid cell compartment. Cell subset structures previously defined by protein marker expression need to be revised based on unbiased analyses of scRNA-seq data. Particularly in inflammatory conditions, myeloid cells exhibit substantially vaster heterogeneity than previously anticipated, and work performed within large international projects, such as the Human Cell Atlas, has already revealed novel tissue macrophage subsets. Based on these exciting developments, we propose the next steps to a full understanding of the myeloid cell compartment in health and diseases.

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 pathophysiology of sepsis and precision-medicine-based immunotherapy.

Sepsis remains a major cause of morbidity and mortality in both low- and high-income countries. Antibiotic therapy and supportive care have significantly improved survival following sepsis in the twentieth century, but further progress has been challenging. Immunotherapy trials for sepsis, mainly aimed at suppressing the immune response, from the 1990s and 2000s, have largely failed, in part owing to unresolved patient heterogeneity in the underlying immune disbalance. The past decade has brought the promise to break this blockade through technological developments based on omics-based technologies and systems medicine that can provide a much larger data space to describe in greater detail the immune endotypes in sepsis. Patient stratification opens new avenues towards precision medicine approaches that aim to apply immunotherapies to sepsis, on the basis of precise biomarkers and molecular mechanisms defining specific immune endotypes. This approach has the potential to lead to the establishment of immunotherapy as a successful pillar in the treatment of sepsis for future generations.

COVID-19 and the human innate immune system.

The introduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the human population represents a tremendous medical and economic crisis. Innate immunity-as the first line of defense of our immune system-plays a central role in combating this novel virus. Here, we provide a conceptual framework for the interaction of the human innate immune system with SARS-CoV-2 to link the clinical observations with experimental findings that have been made during the first year of the pandemic. We review evidence that variability in innate immune system components among humans is a main contributor to the heterogeneous disease courses observed for coronavirus disease 2019 (COVID-19), the disease spectrum induced by SARS-CoV-2. A better understanding of the pathophysiological mechanisms observed for cells and soluble mediators involved in innate immunity is a prerequisite for the development of diagnostic markers and therapeutic strategies targeting COVID-19. However, this will also require additional studies addressing causality of events, which so far are lagging behind.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

A guide to systems-level immunomics.

The immune system is highly complex and distributed throughout an organism, with hundreds to thousands of cell states existing in parallel with diverse molecular pathways interacting in a highly dynamic and coordinated fashion. Although the characterization of individual genes and molecules is of the utmost importance for understanding immune-system function, high-throughput, high-resolution omics technologies combined with sophisticated computational modeling and machine-learning approaches are creating opportunities to complement standard immunological methods with new insights into immune-system dynamics. Like systems immunology itself, immunology researchers must take advantage of these technologies and form their own diverse networks, connecting with researchers from other disciplines. This Review is an introduction and 'how-to guide' for immunologists with no particular experience in the field of omics but with the intention to learn about and apply these systems-level approaches, and for immunologists who want to make the most of interdisciplinary networks.

Urban living in healthy Tanzanians is associated with an inflammatory status driven by dietary and metabolic changes.

Sub-Saharan Africa currently experiences an unprecedented wave of urbanization, which has important consequences for health and disease patterns. This study aimed to investigate and integrate the immune and metabolic consequences of rural or urban lifestyles and the role of nutritional changes associated with urban living. In a cohort of 323 healthy Tanzanians, urban as compared to rural living was associated with a pro-inflammatory immune phenotype, both at the transcript and protein levels. We identified different food-derived and endogenous circulating metabolites accounting for these differences. Serum from urban dwellers induced reprogramming of innate immune cells with higher tumor necrosis factor production upon microbial re-stimulation in an in vitro model of trained immunity. These data demonstrate important shifts toward an inflammatory phenotype associated with an urban lifestyle and provide new insights into the underlying dietary and metabolic factors, which may affect disease epidemiology in sub-Sahara African countries.

CRELD1 modulates homeostasis of the immune system in mice and humans.

CRELD1 is a pivotal factor for heart development, the function of which is unknown in adult life. We here provide evidence that CRELD1 is an important gatekeeper of immune system homeostasis. Exploiting expression variance in large human cohorts contrasting individuals with the lowest and highest CRELD1 expression levels revealed strong phenotypic, functional and transcriptional differences, including reduced CD4+ T cell numbers. These findings were validated in T cell-specific Creld1-deficient mice. Loss of Creld1 was associated with simultaneous overactivation and increased apoptosis, resulting in a net loss of T cells with age. Creld1 was transcriptionally and functionally linked to Wnt signaling. Collectively, gene expression variance in large human cohorts combined with murine genetic models, transcriptomics and functional testing defines CRELD1 as an important modulator of immune homeostasis.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

Whole blood stimulation as a tool for studying the human immune system.

The human immune system is best accessible via tissues and organs not requiring major surgical intervention, such as blood. In many circumstances, circulating immune cells correlate with an individual's health state and give insight into physiological and pathophysiological processes. Stimulating whole blood ex vivo is a powerful tool to investigate immune responses. In the context of clinical research, the applications of whole blood stimulation include host immunity, disease characterization, diagnosis, treatment, and drug development. Here, we summarize different setups and readouts of whole blood assays and discuss applications for preclinical research and clinical practice. Finally, we propose combining whole blood stimulation with high-throughput technologies, such as single-cell RNA-sequencing, to comprehensively analyze the human immune system for the identification of biomarkers, therapeutic interventions as well as companion diagnostics.

Molecular mechanisms and treatment responses of pulmonary fibrosis in severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients can develop pulmonary fibrosis (PF), which is associated with impaired outcome. We assessed specific leukocytic transcriptome profiles associated with PF and the influence of early dexamethasone (DEXA) treatment on the clinical course of PF in critically ill COVID-19 patients. METHODS: We performed a pre-post design study in 191 COVID-19 patients admitted to the Intensive Care Unit (ICU) spanning two treatment cohorts: the pre-DEXA- (n = 67) and the DEXA-cohort (n = 124). PF was identified based on radiological findings, worsening of ventilatory parameters and elevated circulating PIIINP levels. Longitudinal transcriptome profiles of 52 pre-DEXA patients were determined using RNA sequencing. Effects of prednisone treatment on clinical fibrosis parameters and outcomes were analyzed between PF- and no-PF-patients within both cohorts. RESULTS: Transcriptome analyses revealed upregulation of inflammatory, coagulation and neutrophil extracellular trap-related pathways in PF-patients compared to no-PF patients. Key genes involved included PADI4, PDE4D, MMP8, CRISP3, and BCL2L15. Enrichment of several identified pathways was associated with impaired survival in a external cohort of patients with idiopathic pulmonary fibrosis. Following prednisone treatment, PF-related profiles reverted towards those observed in the no-PF-group. Likewise, PIIINP levels decreased significantly following prednisone treatment. PF incidence was 28% and 25% in the pre-DEXA- and DEXA-cohort, respectively (p = 0.61). ICU length-of-stay (pre-DEXA: 42 [29-49] vs. 18 [13-27] days, p < 0.001; DEXA: 42 [28-57] vs. 13 [7-24] days, p < 0.001) and mortality (pre-DEXA: 47% vs. 15%, p = 0.009; DEXA: 61% vs. 19%, p < 0.001) were higher in the PF-groups compared to the no-PF-groups within both cohorts. Early dexamethasone therapy did not influence these outcomes. CONCLUSIONS: ICU patients with COVID-19 who develop PF exhibit upregulated coagulation, inflammation, and neutrophil extracellular trap-related pathways as well as prolonged ICU length-of-stay and mortality. This study indicates that early dexamethasone treatment neither influences the incidence or clinical course of PF, nor clinical outcomes.

NCX1 represents an ionic Na+ sensing mechanism in macrophages.

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

Creld1 regulates myocardial development and function.

CRELD1 (Cysteine-Rich with EGF-Like Domains 1) is a risk gene for non-syndromic atrioventricular septal defects in human patients. In a mouse model, Creld1 has been shown to be essential for heart development, particularly in septum and valve formation. However, due to the embryonic lethality of global Creld1 knockout (KO) mice, its cell type-specific function during peri- and postnatal stages remains unknown. Here, we generated conditional Creld1 KO mice lacking Creld1 either in the endocardium (KOTie2) or the myocardium (KOMyHC). Using a combination of cardiac phenotyping, histology, immunohistochemistry, RNA-sequencing, and flow cytometry, we demonstrate that Creld1 function in the endocardium is dispensable for heart development. Lack of myocardial Creld1 causes extracellular matrix remodeling and trabeculation defects by modulation of the Notch1 signaling pathway. Hence, KOMyHC mice die early postnatally due to myocardial hypoplasia. Our results reveal that Creld1 not only controls the formation of septa and valves at an early stage during heart development, but also cardiac maturation and function at a later stage. These findings underline the central role of Creld1 in mammalian heart development and function.

Systems immunology allows a new view on human dendritic cells.

As the most important antigen-presenting cells, dendritic cells connect the innate and adaptive part of our immune system and play a pivotal role in our course of action against invading pathogens as well as during successful vaccination. Immunologists have therefore studied these cells in great detail using flow cytometry-based analyses, in vitro assays and in vivo models, both in murine models and in humans. Albeit, sophisticated, classical immunological, and molecular approaches were often unable to unequivocally determine the subpopulation structure of the dendritic cell lineage and not surprisingly, conflicting results about dendritic cell subsets co-existed throughout the last decades. With the advent of systems approaches and the most recent introduction of -omics approaches on the single cell level combined with multi-colour flow cytometry or mass cytometry, we now enter an era allowing us to define cell population structures with an unprecedented precision. We will report here on the most recent studies applying these technologies to human dendritic cells. Proper delineation of and definition of molecular signatures for the different human dendritic cell subsets will greatly facilitate studying these cells in the future: understanding their function under physiological as well as pathological conditions.

Modeling population heterogeneity from microbial communities to immune response in cells.

Heterogeneity is universally observed in all natural systems and across multiple scales. Understanding population heterogeneity is an intriguing and attractive topic of research in different disciplines, including microbiology and immunology. Microbes and mammalian immune cells present obviously rather different system-specific biological features. Nevertheless, as typically occurs in science, similar methods can be used to study both types of cells. This is particularly true for mathematical modeling, in which key features of a system are translated into algorithms to challenge our mechanistic understanding of the underlying biology. In this review, we first present a broad overview of the experimental developments that allowed observing heterogeneity at the single cell level. We then highlight how this "data revolution" requires the parallel advancement of algorithms and computing infrastructure for data processing and analysis, and finally present representative examples of computational models of population heterogeneity, from microbial communities to immune response in cells.

Neue Krankheiten mit Bluttranskriptomik entschlüsseln.

The COVID-19 pandemic is leading to increasing numbers of patients all over the world. Reports on a dysregulated immune system in the severe cases calls for a better characterization of the ongoing changes. To dissect COVID-19-driven immune host responses, we profiled whole blood transcriptomes enabling a data-driven stratification based on molecular phenotype. This analysis allowed prediction of patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host.

New "programmers" in tissue macrophage activation.

Tissue macrophages and monocyte-derived macrophages are under continuous influence from environmental signals that define their activation status. Along these lines, macrophages integrate tissue and stress signals and are specifically programmed by these signals towards a spectrum of functions necessary to fulfill their duty within their particular microenvironment, be it homeostatic tissue function, response to inflammatory pathophysiology, or even resolution of an inflammation. Recent years have seen tremendous progress in our understanding how macrophages at different sites are transcriptionally and epigenetically programmed to execute their diverse tasks throughout the body. The identification of transcription factors guiding these reprogramming activities is currently a major topic in macrophage research. We summarize the most recent findings within the last 18 months concerning the identification of novel transcription factors associated with particular macrophage location or function. Furthermore, we extend the view of cellular programming of macrophages to additional levels of regulation, for example, by long non-coding RNAs. Clearly, in addition to transcription factors, there are many more "programmers" shaping the versatile functionality of these exciting innate immune cells.

Complementation in trans of altered thymocyte development in mice expressing mutant forms of the adaptor molecule SLP76.

The adaptor protein SLP76 directs signaling downstream of the T cell receptor (TCR) and is essential for thymocyte development. SLP76 contains three N-terminal tyrosines that are critical for its function. To define the role of these residues in thymocyte development, we generated two lines of "knock-in" mice, one expressing a mutation in tyrosine 145 (Y145F) and a second harboring two point mutations at tyrosines 112 and 128 (Y112-128F). We show here that although thymocyte development requires both Y145- and Y112-128-generated signals, selection was more dependent upon Y145. Although several proximal TCR signaling events were defective in both mutant mice, phosphorylation of the guanine nucleotide exchange factor, Vav1, and activation of Itk-dependent pathways were differentially affected by mutations at Y112-128 and Y145, respectively. Analysis of mice expressing one Y145F and one Y112-128F allele revealed that these mutants could complement one another in trans, demonstrating cooperativity between two or more SLP76 molecules. Thus, the N-terminal tyrosines of SLP76 are required for thymocyte selection but can function on separate molecules to support TCR signaling.