Multiomic analyses uncover immunological signatures in acute and chronic coronary syndromes.

Acute and chronic coronary syndromes (ACS and CCS) are leading causes of mortality. Inflammation is considered a key pathogenic driver of these diseases, but the underlying immune states and their clinical implications remain poorly understood. Multiomic factor analysis (MOFA) allows unsupervised data exploration across multiple data types, identifying major axes of variation and associating these with underlying molecular processes. We hypothesized that applying MOFA to multiomic data obtained from blood might uncover hidden sources of variance and provide pathophysiological insights linked to clinical needs. Here we compile a longitudinal multiomic dataset of the systemic immune landscape in both ACS and CCS (n = 62 patients in total, n = 15 women and n = 47 men) and validate this in an external cohort (n = 55 patients in total, n = 11 women and n = 44 men). MOFA reveals multicellular immune signatures characterized by distinct monocyte, natural killer and T cell substates and immune-communication pathways that explain a large proportion of inter-patient variance. We also identify specific factors that reflect disease state or associate with treatment outcome in ACS as measured using left ventricular ejection fraction. Hence, this study provides proof-of-concept evidence for the ability of MOFA to uncover multicellular immune programs in cardiovascular disease, opening new directions for mechanistic, biomarker and therapeutic studies.

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Peripheral priming induces plastic transcriptomic and proteomic responses in circulating neutrophils required for pathogen containment.

Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow are shaped within the circulation remains vague. Experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile human and murine neutrophil transcriptomes and proteomes during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to dynamic shifts dominated by conserved up-regulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB signaling-dependent up-regulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by up-regulation of distinct effector molecules upon infection. These data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation.

Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection.

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.

Self-sustaining IL-8 loops drive a prothrombotic neutrophil phenotype in severe COVID-19.

Neutrophils provide a critical line of defense in immune responses to various pathogens, inflicting self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients with coronavirus disease 2019 (COVID-19). Here, we use state-of-the art mass spectrometry-based proteomics and transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic IL-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces severe acute respiratory distress syndrome of coronavirus 2 (SARS-CoV-2) spike protein-induced, human ACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8 axis as a promising therapeutic target in severe SARS-CoV-2 infection.

Vascular neutrophilic inflammation and immunothrombosis distinguish severe COVID-19 from influenza pneumonia.

OBJECTIVE: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe pneumonia, but also thrombotic complications and non-pulmonary organ failure. Recent studies suggest intravascular neutrophil activation and subsequent immune cell-triggered immunothrombosis as a central pathomechanism linking the heterogenous clinical picture of coronavirus disease 2019 (COVID-19). We sought to study whether immunothrombosis is a pathognomonic factor in COVID-19 or a general feature of (viral) pneumonia, as well as to better understand its upstream regulation. APPROACH AND RESULTS: By comparing histopathological specimens of SARS-CoV-2 with influenza-affected lungs, we show that vascular neutrophil recruitment, NETosis, and subsequent immunothrombosis are typical features of severe COVID-19, but less prominent in influenza pneumonia. Activated neutrophils were typically found in physical association with monocytes. To explore this further, we combined clinical data of COVID-19 cases with comprehensive immune cell phenotyping and bronchoalveolar lavage fluid scRNA-seq data. We show that a HLADRlow CD9low monocyte population expands in severe COVID-19, which releases neutrophil chemokines in the lungs, and might in turn explain neutrophil expansion and pulmonary recruitment in the late stages of severe COVID-19. CONCLUSIONS: Our data underline an innate immune cell axis causing vascular inflammation and immunothrombosis in severe SARS-CoV-2 infection.

SEQU-INTO: Early detection of impurities, contamination and off-targets (ICOs) in long read/MinION sequencing.

The MinION sequencer by Oxford Nanopore Technologies turns DNA and RNA sequencing into a routine task in biology laboratories or in field research. For downstream analysis it is required to have a sufficient amount of target reads. Especially prokaryotic or bacteriophagic sequencing samples can contain a significant amount of off-target sequences in the processed sample, stemming from human DNA/RNA contamination, insufficient rRNA depletion, or remaining DNA/RNA from other organisms (e.g. host organism from bacteriophage cultivation). Such impurity, contamination and off-targets (ICOs) block read capacity, requiring to sequence deeper. In comparison to second-generation sequencing, MinION sequencing allows to reuse its chip after a (partial) run. This allows further usage of the same chip with more sample, even after adjusting the library preparation to reduce ICOs. The earlier a sample's ICOs are detected, the better the sequencing chip can be conserved for future use. Here we present sequ-into, a low-resource and user-friendly cross-platform tool to detect ICO sequences from a predefined ICO database in samples early during a MinION sequencing run. The data provided by sequ-into empowers the user to quickly take action to preserve sample material and chip capacity. sequ-into is available from https://github.com/mjoppich/sequ-into.

From command-line bioinformatics to bioGUI.

Bioinformatics is a highly interdisciplinary field providing (bioinformatics) applications for scientists from many disciplines. Installing and starting applications on the command-line (CL) is inconvenient and/or inefficient for many scientists. Nonetheless, most methods are implemented with a command-line interface only. Providing a graphical user interface (GUI) for bioinformatics applications is one step toward routinely making CL-only applications available to more scientists and, thus, toward a more effective interdisciplinary work. With our bioGUI framework we address two main problems of using CL bioinformatics applications: First, many tools work on UNIX-systems only, while many scientists use Microsoft Windows. Second, scientists refrain from using CL tools which, however, could well support them in their research. With bioGUI install modules and templates, installing and using CL tools is made possible for most scientists-even on Windows, due to bioGUI's support for Windows Subsystem for Linux. In addition, bioGUI templates can easily be created, making the bioGUI framework highly rewarding for developers. From the bioGUI repository it is possible to download, install and use bioinformatics tools with just a few clicks.

Using Context-Sensitive Text Mining to Identify miRNAs in Different Stages of Atherosclerosis.

790 human and mouse micro-RNAs (miRNAs) are involved in diseases. More than 26,428 miRNA-gene interactions are annotated in humans and mice. Most of these interactions are posttranscriptional regulations: miRNAs bind to the messenger RNAs (mRNAs) of genes and induce their degradation, thereby reducing the gene expression of target genes. For atherosclerosis, 667 miRNA-gene interactions for 124 miRNAs and 343 genes have been identified and described in numerous publications. Some interactions were observed through high-throughput experiments, others were predicted using bioinformatic methods, and some were determined by targeted experiments. Several reviews collect knowledge on miRNA-gene interactions in (specific aspects of) atherosclerosis.Here, we use our bioinformatics resource (atheMir) to give an overview of miRNA-gene interactions in the context of atherosclerosis. The interactions are based on public databases and context-based text mining of 28 million PubMed abstracts. The miRNA-gene interactions are obtained from more than 10,000 publications, of which more than 1,000 are in a cardiovascular disease context (266 in atherosclerosis). We discuss interesting miRNA-gene interactions in atherosclerosis, grouped by specific processes in different cell types and six phases of atherosclerotic progression. All evidence is referenced and easily accessible: Relevant interactions are provided by atheMir as supplementary tables for further evaluation and, for example, for the subsequent data analysis of high-throughput measurements as well as for the generation and validation of hypotheses. The atheMir approach has several advantages: (1) the evidence is easily accessible, (2) regulatory interactions are uniformly available for subsequent high-throughput data analysis, and (3) the resource can incrementally be updated with new findings.

Tryptophan usage by Helicobacter pylori differs among strains.

Because of its association with severe gastric pathologies, including gastric cancer, Helicobacter pylori has been subject of research for more than 30 years. Its capacity to adapt and survive in the human stomach can be attributed to its genetic flexibility. Its natural competence and its capacity to turn genes on and off allows H. pylori to adapt rapidly to the changing conditions of its host. Because of its genetic variability, it is difficult to establish the uniqueness of each strain obtained from a human host. The methods considered to-date to deliver the best result for differentiation of strains are Rapid Amplification of Polymorphic DNA (RAPD), Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) analysis. While RAPD analysis is cost-effective, it requires a stable genome for its reliability. MLST and WGS are optimal for strain identification, however, they require analysis of data at the bioinformatics level. Using the StainFree method, which modifies tryptophan residues on proteins using 2, 2, 2, - trichloroethanol (TCE), we observed a strain specific pattern of tryptophan in 1D acrylamide gels. In order to establish the effectiveness of tryptophan fingerprinting for strain identification, we compared the graphic analysis of tryptophan-labelled bands in the gel images with MLST results. Based on this, we find that tryptophan banding patterns can be used as an alternative method for the differentiation of H. pylori strains. Furthermore, investigating the origin for these differences, we found that H. pylori strains alters the number and/or position of tryptophan present in several proteins at the genetic code level, with most exchanges taking place in membrane- and cation-binding proteins, which could be part of a novel response of H. pylori to host adaptation.