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.

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.

Identification of drug candidates targeting monocyte reprogramming in people living with HIV.

Introduction: People living with HIV (PLHIV) are characterized by functional reprogramming of innate immune cells even after long-term antiretroviral therapy (ART). In order to assess technical feasibility of omics technologies for application to larger cohorts, we compared multiple omics data layers. Methods: Bulk and single-cell transcriptomics, flow cytometry, proteomics, chromatin landscape analysis by ATAC-seq as well as ex vivo drug stimulation were performed in a small number of blood samples derived from PLHIV and healthy controls from the 200-HIV cohort study. Results: Single-cell RNA-seq analysis revealed that most immune cells in peripheral blood of PLHIV are altered in their transcriptomes and that a specific functional monocyte state previously described in acute HIV infection is still existing in PLHIV while other monocyte cell states are only occurring acute infection. Further, a reverse transcriptome approach on a rather small number of PLHIV was sufficient to identify drug candidates for reversing the transcriptional phenotype of monocytes in PLHIV. Discussion: These scientific findings and technological advancements for clinical application of single-cell transcriptomics form the basis for the larger 2000-HIV multicenter cohort study on PLHIV, for which a combination of bulk and single-cell transcriptomics will be included as the leading technology to determine disease endotypes in PLHIV and to predict disease trajectories and outcomes.

Chromatin accessibility profiling of targeted cell populations with laser capture microdissection coupled to ATAC-seq.

Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers.

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.

Fighting Post-COVID and ME/CFS - development of curative therapies.

The sequela of COVID-19 include a broad spectrum of symptoms that fall under the umbrella term post-COVID-19 condition or syndrome (PCS). Immune dysregulation, autoimmunity, endothelial dysfunction, viral persistence, and viral reactivation have been identified as potential mechanisms. However, there is heterogeneity in expression of biomarkers, and it is unknown yet whether these distinguish different clinical subgroups of PCS. There is an overlap of symptoms and pathomechanisms of PCS with postinfectious myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). No curative therapies are available for ME/CFS or PCS. The mechanisms identified so far provide targets for therapeutic interventions. To accelerate the development of therapies, we propose evaluating drugs targeting different mechanisms in clinical trial networks using harmonized diagnostic and outcome criteria and subgrouping patients based on a thorough clinical profiling including a comprehensive diagnostic and biomarker phenotyping.

huva: A human variation analysis framework to predict gene perturbation from population-scale multi-omics data.

Variance of gene expression is intrinsic to any given natural population. Here, we present a protocol to analyze this variance using a conditional quasi loss- and gain-of-function approach. The huva (human variation) package takes advantage of population-scale multi-omics data to infer gene function and the relationship between phenotype and gene expression. We describe the steps for setting up the huva workspace, formatting datasets, performing huva experiments, and exporting data. For complete details on the use and execution of this protocol, please refer to Bonaguro et al. (2022).1.

Human variation in population-wide gene expression data predicts gene perturbation phenotype.

Population-scale datasets of healthy individuals capture genetic and environmental factors influencing gene expression. The expression variance of a gene of interest (GOI) can be exploited to set up a quasi loss- or gain-of-function "in population" experiment. We describe here an approach, huva (human variation), taking advantage of population-scale multi-layered data to infer gene function and relationships between phenotypes and expression. Within a reference dataset, huva derives two experimental groups with LOW or HIGH expression of the GOI, enabling the subsequent comparison of their transcriptional profile and functional parameters. We demonstrate that this approach robustly identifies the phenotypic relevance of a GOI allowing the stratification of genes according to biological functions, and we generalize this concept to almost 16,000 genes in the human transcriptome. Additionally, we describe how huva predicts monocytes to be the major cell type in the pathophysiology of STAT1 mutations, evidence validated in a clinical cohort.

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.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Decoding mechanism of action and sensitivity to drug candidates from integrated transcriptome and chromatin state.

Omics-based technologies are driving major advances in precision medicine, but efforts are still required to consolidate their use in drug discovery. In this work, we exemplify the use of multi-omics to support the development of 3-chloropiperidines, a new class of candidate anticancer agents. Combined analyses of transcriptome and chromatin accessibility elucidated the mechanisms underlying sensitivity to test agents. Furthermore, we implemented a new versatile strategy for the integration of RNA- and ATAC-seq (Assay for Transposase-Accessible Chromatin) data, able to accelerate and extend the standalone analyses of distinct omic layers. This platform guided the construction of a perturbation-informed basal signature predicting cancer cell lines' sensitivity and to further direct compound development against specific tumor types. Overall, this approach offers a scalable pipeline to support the early phases of drug discovery, understanding of mechanisms, and potentially inform the positioning of therapeutics in the clinic.

Mature neutrophils and a NF-κB-to-IFN transition determine the unifying disease recovery dynamics in COVID-19.

Disease recovery dynamics are often difficult to assess, as patients display heterogeneous recovery courses. To model recovery dynamics, exemplified by severe COVID-19, we apply a computational scheme on longitudinally sampled blood transcriptomes, generating recovery states, which we then link to cellular and molecular mechanisms, presenting a framework for studying the kinetics of recovery compared with non-recovery over time and long-term effects of the disease. Specifically, a decrease in mature neutrophils is the strongest cellular effect during recovery, with direct implications on disease outcome. Furthermore, we present strong indications for global regulatory changes in gene programs, decoupled from cell compositional changes, including an early rise in T cell activation and differentiation, resulting in immune rebalancing between interferon and NF-κB activity and restoration of cell homeostasis. Overall, we present a clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues.

The 2000HIV study: Design, multi-omics methods and participant characteristics.

Background: Even during long-term combination antiretroviral therapy (cART), people living with HIV (PLHIV) have a dysregulated immune system, characterized by persistent immune activation, accelerated immune ageing and increased risk of non-AIDS comorbidities. A multi-omics approach is applied to a large cohort of PLHIV to understand pathways underlying these dysregulations in order to identify new biomarkers and novel genetically validated therapeutic drugs targets. Methods: The 2000HIV study is a prospective longitudinal cohort study of PLHIV on cART. In addition, untreated HIV spontaneous controllers were recruited. In-depth multi-omics characterization will be performed, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and metagenomics, functional immunological assays and extensive immunophenotyping. Furthermore, the latent viral reservoir will be assessed through cell associated HIV-1 RNA and DNA, and full-length individual proviral sequencing on a subset. Clinical measurements include an ECG, carotid intima-media thickness and plaque measurement, hepatic steatosis and fibrosis measurement as well as psychological symptoms and recreational drug questionnaires. Additionally, considering the developing pandemic, COVID-19 history and vaccination was recorded. Participants return for a two-year follow-up visit. The 2000HIV study consists of a discovery and validation cohort collected at separate sites to immediately validate any finding in an independent cohort. Results: Overall, 1895 PLHIV from four sites were included for analysis, 1559 in the discovery and 336 in the validation cohort. The study population was representative of a Western European HIV population, including 288 (15.2%) cis-women, 463 (24.4%) non-whites, and 1360 (71.8%) MSM (Men who have Sex with Men). Extreme phenotypes included 114 spontaneous controllers, 81 rapid progressors and 162 immunological non-responders. According to the Framingham score 321 (16.9%) had a cardiovascular risk of >20% in the next 10 years. COVID-19 infection was documented in 234 (12.3%) participants and 474 (25.0%) individuals had received a COVID-19 vaccine. Conclusion: The 2000HIV study established a cohort of 1895 PLHIV that employs multi-omics to discover new biological pathways and biomarkers to unravel non-AIDS comorbidities, extreme phenotypes and the latent viral reservoir that impact the health of PLHIV. The ultimate goal is to contribute to a more personalized approach to the best standard of care and a potential cure for PLHIV.

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.

Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs.

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5-E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

Alveolar macrophage transcriptomic profiling in COPD shows major lipid metabolism changes.

BACKGROUND: Immune cells play a major role in the pathogenesis of COPD. Changes in the distribution and cellular functions of major immune cells, such as alveolar macrophages (AMs) and neutrophils are well known; however, their transcriptional reprogramming and contribution to the pathophysiology of COPD are still not fully understood. METHOD: To determine changes in transcriptional reprogramming and lipid metabolism in the major immune cell type within bronchoalveolar lavage fluid, we analysed whole transcriptomes and lipidomes of sorted CD45+Lin-HLA-DR+CD66b-Autofluorescencehi AMs from controls and COPD patients. RESULTS: We observed global transcriptional reprogramming featuring a spectrum of activation states, including pro- and anti-inflammatory signatures. We further detected significant changes between COPD patients and controls in genes involved in lipid metabolism, such as fatty acid biosynthesis in GOLD2 patients. Based on these findings, assessment of a total of 202 lipid species in sorted AMs revealed changes of cholesteryl esters, monoacylglycerols and phospholipids in a disease grade-dependent manner. CONCLUSIONS: Transcriptome and lipidome profiling of COPD AMs revealed GOLD grade-dependent changes, such as in cholesterol metabolism and interferon-α and γ responses.

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.

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.

Neutrophils in COVID-19.

Strong evidence has been accumulated since the beginning of the COVID-19 pandemic that neutrophils play an important role in the pathophysiology, particularly in those with severe disease courses. While originally considered to be a rather homogeneous cell type, recent attention to neutrophils has uncovered their fascinating transcriptional and functional diversity as well as their developmental trajectories. These new findings are important to better understand the many facets of neutrophil involvement not only in COVID-19 but also many other acute or chronic inflammatory diseases, both communicable and non-communicable. Here, we highlight the observed immune deviation of neutrophils in COVID-19 and summarize several promising therapeutic attempts to precisely target neutrophils and their reactivity in patients with COVID-19.

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.