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.

Thrombocytopenia and splenic platelet-directed immune responses after IV ChAdOx1 nCov-19 administration.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are based on a range of novel platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently, a novel complication of SARS-CoV-2-targeted adenovirus vaccines has emerged: immune thrombocytopenia, either isolated, or accompanied by thrombosis (then termed VITT). This complication is characterized by low platelet counts, and in the case of VITT, also by platelet-activating platelet factor 4 antibodies reminiscent of heparin-induced thrombocytopenia, leading to a prothrombotic state with clot formation at unusual anatomic sites. Here, we detected antiplatelet antibodies targeting platelet glycoprotein receptors in 30% of patients with proven VITT (n = 27) and 42% of patients with isolated thrombocytopenia after ChAdOx1 nCov-19 vaccination (n = 26), indicating broad antiplatelet autoimmunity in these clinical entities. We use in vitro and in vivo models to characterize possible mechanisms of these platelet-targeted autoimmune responses leading to thrombocytopenia. We show that IV but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation in mice. After IV injection, these aggregates are phagocytosed by macrophages in the spleen, and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of platelet-associated complications after ChAdOx1 nCov-19 administration and highlights accidental IV injection as a potential mechanism of platelet-targeted autoimmunity. Hence, preventing IV injection when administering adenovirus-based vaccines could be a potential measure against platelet-associated pathologies after vaccination.

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.

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.

Differential Effects of Erythropoietin Administration and Overexpression on Venous Thrombosis in Mice.

BACKGROUND: Deep vein thrombosis (DVT) is a common condition associated with significant mortality due to pulmonary embolism. Despite advanced prevention and anticoagulation therapy, the incidence of venous thromboembolism remains unchanged. Individuals with elevated hematocrit and/or excessively high erythropoietin (EPO) serum levels are particularly susceptible to DVT formation. We investigated the influence of short-term EPO administration compared to chronic EPO overproduction on DVT development. Additionally, we examined the role of the spleen in this context and assessed its impact on thrombus composition. METHODS: We induced ligation of the caudal vena cava (VCC) in EPO-overproducing Tg(EPO) mice as well as wildtype mice treated with EPO for two weeks, both with and without splenectomy. The effect on platelet circulation time was evaluated through FACS analysis, and thrombus composition was analyzed using immunohistology. RESULTS: We present evidence for an elevated thrombogenic phenotype resulting from chronic EPO overproduction, achieved by combining an EPO-overexpressing mouse model with experimental DVT induction. This increased thrombotic state is largely independent of traditional contributors to DVT, such as neutrophils and platelets. Notably, the pronounced prothrombotic effect of red blood cells (RBCs) only manifests during chronic EPO overproduction and is not influenced by splenic RBC clearance, as demonstrated by splenectomy. In contrast, short-term EPO treatment does not induce thrombogenesis in mice. Consequently, our findings support the existence of a differential thrombogenic effect between chronic enhanced erythropoiesis and exogenous EPO administration. CONCLUSION: Chronic EPO overproduction significantly increases the risk of DVT, while short-term EPO treatment does not. These findings underscore the importance of considering EPO-related factors in DVT risk assessment and potential therapeutic strategies.

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.

SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast.

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast  Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong  adh1 promoter. To overcome these problems we have developed an improved system,  SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for  S. pombe expressed from the medium strength  adh15 promoter. The  SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the  BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The  SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the  ade6 +  or  ura4 + genes by transformation of cells from asynchronous cultures.  SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the  SpEDIT system displayed reduced toxicity compared to currently available  S. pombe editing systems. Thus,  SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.