Tissue-resident macrophages receive signals from their microenvironment that coordinate the activity of transcription factors (TFs) to establish distinct transcriptional profiles and identities. However, the molecular mechanisms whereby interactions with other cells of the niche imprint a distinct macrophage identity remain poorly understood. Here, we report that retinoid X receptors (RXRs) determine the differentiation and identity of alveolar macrophages (AM) by regulating chromatin accessibility and transcriptional activity of AM-core and function genes, enabling PPARγ-dependent programs. AM differentiation and maintenance in vivo require RXR upregulation in response to tissue-derived δ-like canonical Notch ligand 4 (DLL4), GM-CSF, and TGF β (TGFβ). Interplay among these signals leads to cooperation between RXRα, RBPJ, STAT5, and SMAD4 for the transcriptional and epigenetic regulation of key AM-core genes. These results underscore the role of RXRs as key TFs that cooperate with other regulatory elements to establish the AM population and determine AM identity.

Despite intriguing roles for the Succinate receptor (Sucnr1) in inflammation, few studies have explored its role in hematopoiesis. Here, we show that low SUCNR1 represents a marker for reduced overall and progression-free survival in acute myeloid leukemia (AML) patients. Succinic acid, which displays Sucnr1-dependent and independent effects, promotes disease in mouse models of pre-leukemic myelopoiesis, AML and AML xenografts, expressing low SUCNR1. In vivo global or hematopoietic deletion of Sucnr1 induces expansion of hematopoietic stem and progenitor cells (HSPC) and hematopoiesis, whilst Sucnr1-tomato+ HSPC display restricted engraftment potential. Mechanistically, activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC and preserves HSPC transcriptional programs via control of S100a8/S100a9. Blocking S100a9 with tasquinimod rescues the defects of Sucnr1 knock-out mice, and combined with a potent Sucnr1 agonist shows therapeutic value in AML mice. In AML xenografts, single-cell RNA-sequencing reanalyses confirm SUCNR1 as a therapeutic vulnerability in patients. Together, Sucnr1 signaling restricts hematopoiesis at least partially through HSPC and via control of S100a8/S100a9. Its dysregulation emerges as contributor to malignancy that opens therapeutic avenues for AML patients.

BackgroundRecent advances in instrument sensitivity and sample preparation techniques are significantly improving the ability to study the heterogeneity of cell populations at the single-cell level by mass spectrometry-based proteomics. Integrating multiple layers of cellular data offers additional and yet unexplored insights in single-cell proteomics. In regenerative research, cardiomyocyte proliferation driven by the overexpression of the Myc transcription factor has been described, however, it has not yet been investigated at single-cell resolution.ResultsBy using an optimized adult cardiomyocytes isolation procedure from mouse models and taking advantage of the integrative capabilities of the iSanXoT application, we are able to minimize batch effects and cell size-related biases, obtain quantitative subcellular compartment information and detect protein alterations within subcellular compartments, as it had not yet been defined for this methodology. This approach enhances data quantification accuracy and facilitates biological interpretation. We show that the Myc transcription factor switches the expression profile of metabolic enzymes and expands a subpopulation of adult cardiomyocytes with a pro-regenerative signature.ConclusionsWe demonstrate that different layers of information can properly pattern the proteomic phenotype of single-cells. The analysis of single-cardiomyocyte data with the integrative statistical framework of iSanXoT provided important clues to understand the impact of Myc transcription factor in provoking different immaturity and pro-regenerative signatures in adult mouse cardiomyocytes. The cellular heterogeneity exerted by mouse cardiomyocytes upon Myc overexpression demonstrates the relevance of conducting regenerative studies at the single-cell level for precisely defining the amplitude of this response in the heart.

BACKGROUND:Nonmyocytes may contribute to regional adaptive changes during persistent atrial fibrillation (PsAF), favoring its perpetuation. We aimed to investigate the differential features of fibroblast and macrophage populations within individual-specific atrial regions associated with PsAF maintenance. METHODS:The study was conducted in 2 pig models of PsAF with and without infarct-related substrate (N=27 and N=27, respectively) and further validated in humans with PsAF (N=20). Sham-operated pigs (N=9), healthy animals (N=4), and patients in sinus rhythm (N=7) were used as comparative controls. In pigs, in vivo high-density instantaneous frequency modulation maps were used to identify atrial regions associated with PsAF maintenance (drivers). Regional cellular composition and phenotypic states of fibroblast and myeloid lineages were determined using flow cytometry, single-cell RNA sequencing, immunohistochemistry, and proteomic analyses. The functional relevance of driver regions was further studied in patients with symptomatic PsAF undergoing ablation. Flow cytometry and single-cell RNA sequencing analyses were performed in tissue samples of the left atrial appendage in a complementary cohort of patients with PsAF undergoing thoracoscopic-guided ablation. RESULTS:PsAF terminated acutely in 12 of 14 pigs undergoing mapping and ablation of driver regions. In humans, driver ablation was associated with 90% AF-freedom (on/off antiarrhythmic drugs) after 2 years of follow-up. Samples from nonablated pigs revealed a phenotypic shift towards ACTA2 (actin alpha 2)-fibroblasts and PTX3 (pentraxin 3)-fibroblasts during PsAF. Although ACTA2-fibroblasts were highly preserved in human samples, paired comparisons in pig samples showed that PTX3-fibroblasts were enriched only in driver regions. PsAF also showed changes in myeloid cells towards inflammatory profiles. However, regional analysis revealed that, in both humans and pigs with PsAF, driver regions were enriched in cardiac resident macrophages with transcriptomic and proteomic profiles favoring cardiomyocyte homeostasis and cell survival. CONCLUSIONS:PsAF shows differential regional changes in fibroblast and myeloid populations with distinctive gene signatures in areas that drive the overall arrhythmia.

Positron emission tomography (PET) imaging with the radiolabeled glucose analog fluorodeoxyglucose ( 18 FDG) is used to monitor atherosclerosis in clinical trials, but there is uncertainty regarding the plaque cell types that accumulate FDG and how uptake is regulated. The long-standing view that 18 FDG is mainly taken up by macrophages is at odds with human and experimental data, and the impact of disease activity on 18 FDG uptake has not been examined directly. To analyze the ability of 18 FDG-PET to monitor disease activity, we developed a model of plaque regression in minipigs with hepatic overexpression of a gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 ( PCSK9 ). Atherosclerosis was induced through 12 months of high-fat feeding in the porcine model. Disease activity was then lowered for 3 months by reducing plasma cholesterol with a low-fat diet alone or in combination with the microsomal transfer protein (MTP) inhibitor BMS-212122. Plaque regression in advanced lesions of the abdominal aorta was evident from reduced lipid content, reduced necrotic core size, and partial resolution of plaque inflammation and was accompanied by a decline in 18 FDG-PET signal. Single-cell gene expression profiling revealed that plaque regression involved substantial down-regulation of genes encoding glycolytic enzymes in smooth muscle cells (SMCs), macrophages, and lymphocytes, which was corroborated by analysis of the plaque cellular proteome. These findings in a large-animal model suggest that 18 FDG-PET can monitor atherosclerosis because of a close association between disease activity and glycolytic enzyme expression in all of the major plaque cell types.

BACKGROUND:Stroke remains a leading cause of mortality and disability, driven by complex, time-dependent mechanisms that aggravate ischemic injury. Collateral perfusion dictates infarct size, expansion rate, and penumbral preservation, yet its regulation is poorly understood. Beyond structural/genetic factors such as aging or cardiovascular risk, functional influences like circadian immune activity may also affect vascular patency. Neutrophils, key mediators of ischemic injury, exhibit circadian oscillations in phenotype and function that could modulate collateral flow and stroke outcome. METHODS:We combined permanent and transient middle cerebral artery occlusion models in mice with flow cytometry, single-cell RNA sequencing, confocal microscopy, and laser speckle imaging to investigate time-of-day-dependent neutrophil mechanisms in stroke. Pharmacological (chloramidine, DNase-I) and genetic (Pad4 [peptidyl arginine deiminase 4]-/-, Bmal1 [brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1]Neu, Cxcr4 [C-X-C chemokine receptor type 4]Neu) interventions were used to define how time-of-day regulation shapes neutrophil function, neutrophil extracellular traps (NETs) formation, and stroke severity. A cohort of 540 patients with ischemic stroke was analyzed for diurnal patterns of NET-related biomarkers and their association with collateral circulation and clinical outcomes. RESULTS:Infarct volume and neurological deficits exhibited clear circadian oscillations, with worse outcomes when stroke occurred during the murine inactive phase (Zeitgeber time 5) versus the active phase (Zeitgeber time 13). These fluctuations disappeared after neutrophil depletion or clock disruption. During the inactive phase, neutrophils displayed an activated, NET-prone phenotype, causing microvascular stalling and reduced collateral perfusion. Inhibiting NET formation pharmacologically or through Pad4 deletion restored perfusion and abolished time-of-day effects. In patients, neutrophil and NET-related biomarkers (MPO [myeloperoxidase], elastase, sCD40L [soluble CD40 ligand]) showed diurnal oscillations, peaking during the human inactive phase (evening/night), coinciding with reduced collateral flow and poorer outcomes. CONCLUSIONS:Time-of-day regulation of neutrophil function critically determines collateral perfusion and stroke severity. Neutrophil-driven NETosis during the inactive phase promotes microvascular obstruction and worsens outcomes. Targeting NET formation or timing therapy could enhance collateral efficacy and offer novel chronotherapeutic opportunities for stroke treatment.

Stroke is a leading cause of mortality and disability, driven by complex and time-dependent mechanisms that aggravate ischemic damage. Recent evidence indicates that infarct volumes fluctuate according to diurnal oscillations, both in mice and humans, with worse outcomes during the inactive phase of their circadian cycle. Here, we show that neutrophils are responsible for these circadian variations. By depleting neutrophils or blocking their circadian clock, differences in infarct volumes were abolished, suggesting that both the number of neutrophils and their circadian phenotype contribute to ischemic damage. Mechanistically, these differences were linked to the collateral circulation: cerebral blood flow measurements at different times after ischemia showed differential perfusion in the ipsilesional area. We found that, during the inactive mouse phase, neutrophil extracellular traps (NETs), were markedly elevated, coinciding with a reduction in ipsilesional blood flow, a higher percentage of intravascular neutrophils, and an increase in infarct volumes compared with the active phase. These findings underscore a crucial role of neutrophils, their circadian dynamics, and NET release as key drivers of ischemic damage, suggesting novel personalized therapeutic strategies based on circadian rhythms for the treatment of stroke. ### Competing Interest Statement The authors have declared no competing interest.

Most of the mechanistic insights into atrial fibrillation (AF) pathophysiology have been reported on cardiomyocytes, and it is commonly assumed that they apply in the same manner to different regions of the atria. This study aimed to investigate the differential regional features of non-myocyte populations in the atria of pigs and patients with persistent AF (PsAF) based on their functional relevance for AF maintenance. We developed a porcine model resembling clinical PsAF without underlying structural heart disease (N=19), and a second PsAF model with infarct-related atrial cardiomyopathy (MI-PsAF, N=14). Animals with MI-PsAF underwent 3-hour ischaemia-reperfusion in the proximal circumflex artery 2 months prior to the initiation of the AF protocol. The latter aimed to resemble a common clinical scenario with underlying coronary artery disease. After long-lasting self-sustained PsAF, all animals underwent in vivo electroanatomical mapping to identify individual-specific atrial regions associated with AF maintenance (i.e., driver regions), which were further characterized in ex vivo studies by flow cytometry, single-cell RNA transcriptomics (Figure 1), proteomics, and immunolabeling. The results in animals were validated on atrial samples from patients with driver regions in the left atrial appendage (N=8). In a subset of 7 and 6 animals with PsAF and MI-PsAF, respectively, radiofrequency ablation at driver regions effectively terminated the arrhythmia in 100% and 67% of the cases. In patients, ablation of driver regions achieved 90% AF-free survival at 2-years of follow-up. Atrial remodeling during PsAF showed differential adaptative changes in non-myocyte populations, which depended on the functional relevance to sustain the overall arrhythmia. Both in animals and patients, driver regions showed overt compositional shifts in fibroblasts and myeloid populations. More specifically, driver regions were characterized by a phenotypic shift towards cardiac resident macrophages with an associated transcriptomic and proteomic profile favoring cardiomyocyte homeostasis and cell survival within a substrate prone to re-entry (Figure 2). In fibroblasts, PTX3 represented a transcriptional hallmark exclusively present in driver regions, which supports their role on modulating regional specific changes during PsAF. The results reported herein provide a new perspective on the critical role of regional atrial differences in non-myocyte populations that contribute to sustain AF in the long-term.Figure 1 Figure 2

Background and aims:Lipids play a critical role in atherosclerosis. Low-density lipoprotein (LDL)-cholesterol and certain lipid classes like sphingomyelins are associated with inflammation and poor cardiovascular outcomes. Phosphatidylserine (PS), on the other hand, is a negatively charged anti-inflammatory phospholipid class involved in efferocytosis. In this study, we sought to investigate its anti-atherosclerotic properties through a combination of complementary human lipidomics analyses, in vitro assays and in vivo experiments in Apoe -/- mice. Methods:Human lipidomics studies were performed on the 300OB cohort comprising 300 obese and overweight individuals at risk of cardiovascular disease. In vitro assays were carried out using human monocytes and macrophages, and in vivo experiments included histopathological, immunophenotyping and single-cell transcriptomic analyses. Results:In humans, we identified PS as an anti-inflammatory and atheroprotective biomarker. Hence, we developed a high-density lipoprotein (HDL)-like formulation enriched in PS to exploit its properties in a targeted fashion in mice. In vitro , this formulation potently inhibited inflammatory cytokine production on human myeloid cells. Our in-depth in vivo experiments provided evidence of the formulation's potent plaque-stabilizing and anti-inflammatory actions. These effects were mediated by a shift in the monocyte/macrophage compartment toward homeostatic/repairing phenotypes. Conclusions:Collectively, our results demonstrate that HDL-associated PS potently suppresses inflammation and atheroprogression, and holds promise as a viable approach to improve immunomodulatory therapies.

BACKGROUND:Aging is the primary risk factor for atherosclerosis, a degenerative process regulated by immune cells and the leading cause of death worldwide. Previous studies on premature aging syndromes have linked atherosclerosis to defects in A-type lamins, key nuclear envelope components. However, whether these defects influence atherosclerosis during normal aging remains unexplored. Here, we examined how aging affects lamin A/C expression in circulating leukocytes and investigated the impact of manipulating their expression in hematopoietic cells on their function and atherosclerosis progression. METHODS:Flow cytometry assessed lamin A/C expression in human circulating leukocytes. Bone marrow from donor mice was transplanted into lethally irradiated, Ldlr-/--deficient mice to study leukocyte extravasation into the vessel wall via intravital microscopy in the cremaster muscle, and high-fat-diet-induced atherosclerosis via Oil Red O staining of the aorta and carotid arteries. Single-cell RNA sequencing of the aorta was conducted to identify transcriptional changes associated with hematopoietic cell lamin A/C gain-of-function or loss-of-function. RESULTS:Human aging is associated with lower levels of lamin A/C expression in blood-borne leukocytes. To evaluate the functional relationship between hematopoietic lamin A/C expression and atherosclerosis development, we used Lmna-null mice and Lmnatg mice, the latter being the first in vivo model of lamin A gain-of-function. Transplanting lamin A/C-deficient bone marrow into Ldlr-/- mice increased leukocyte extravasation into the vessel wall and accelerated atherosclerosis. Conversely, transplantation of bone marrow overexpressing lamin A into Ldlr-/- receptor mice reduced leukocyte extravasation and atherosclerosis. Single-cell RNA sequencing of atherosclerotic mouse aorta revealed that alterations to hematopoietic cell lamin A/C expression primarily modify the transcriptome of immune cell populations and endothelial cells, affecting their functionality. CONCLUSIONS:We suggest that the age-related decline in lamin A/C expression in blood-borne immune cells contributes to increased leukocyte extravasation and atherosclerosis, highlighting lamin A/C as a novel regulator of age-related atherosclerosis.

Neutrophils exhibit remarkable phenotypic and functional diversity across tissues and diseases1,2, yet the lack of understanding of how this immune compartment is globally organized challenges translation to the clinic. Here we performed single-cell transcriptional profiling of neutrophils spanning 47 anatomical, physiological and pathological scenarios to generate an integrated map of the global neutrophil compartment in mice, which we refer to as NeuMap. NeuMap integrates and expands existing models3,4 to generate fundamental new insights; it reveals that neutrophils organize in a finite number of functional hubs that distribute sequentially during maturation to then branch out into interferon-responsive and immunosuppressive states, as well as a functionally silent state that dominates in the healthy circulation. Computational modelling and timestamp analyses identify prototypical trajectories that connect these hubs, and reveal that the dynamics and preferred paths vary during health, inflammation and cancer. We show that TGFβ, IFNβ and GM-CSF push neutrophils along the different trajectories, and projection of chromatin accessibility sites onto NeuMap reveals that the transcription factor JUNB controls angiogenic and immunosuppressive states and promotes tissue revascularization. The architecture of NeuMap appears to be conserved across sex, environmental and genetic backgrounds, as well as in humans. Finally, we show that NeuMap enables inference of the pathophysiological state of the host by profiling blood neutrophils. Our study delineates the global architecture of the neutrophil compartment and establishes a framework for exploration and exploitation of neutrophil biology.

Eosinophils are leukocytes involved in homeostasis and diseases like asthma. The existence of eosinophil subpopulations remain controversial, with eosinophils being classified as type 1, type 2, inflammatory, resident, homeostatic, or developmental depending on the publication,1 with no clear indication of the existence of intrinsic subpopulations or environmental dependence. Unsupervised methods could provide data on eosinophil true subclassifications, which could be of importance for the efficacy of asthma therapy.2 Therefore, the aim of this study is to analyse blood eosinophils using single-cell RNA sequencing (scRNAseq) (BD Rhapsody™) from three asthmatic subjects and three healthy controls (Table S1). A total of 23,031 isolated individual cells were sequenced, which were mainly annotated as eosinophils (File S1; Figure S1A). Unsupervised clustering yielded 6 clusters (C0–5; Figure 1A; Figure S1). Figure 1A represents individually each study subject, depending on being healthy or asthmatic and in the experiment day (each day an asthmatic and a healthy sample were run in parallel), being batch differences considered in the in-silico analysis. Clusters 0, 1, 2, 3, and 5 were relatively homogeneous, whereas C4 is the more diverse cluster, mainly drifting from the third asthmatic individual (Figure 1A; Figure S1). Gene expression variation between each cluster was low, with C0 having 16 differentially expressed genes, C2 with 2, C3 with 7, and C5 with 4; and only C1 with 41 and C4 with 102 having high numbers (Table S2). As seen in Figure 1B; Figure S2, the most representative genes for each cluster (and between conditions) are genes previously related to eosinophils and asthma (CCR3 and CAT for C1, SP100 and SP110 for C3, CLC and S100A8-9 for C4, or CCL4 and CCL4L2 for C5). Expression of eosinophil markers was conserved between clusters. Interestingly, C1 differentially expressed CCR3, ANXA1, SIGLEC10, and ITGB2, whereas C4 showed higher expression of CLC and SELL (CD62L) (Figure 2A). We applied 3 scores composed of averaged gene expression (Table S3), with clusters having similar scores, even between disease status (Figure 2B), except C4 with higher Th2 asthma score (Figure 2C). Notably, C4 presents the highest transcriptomic variation (fivefold) between healthy and asthmatics (Table S4). EnrichR analysis (Figure 1C; Figure S3) evidenced that C0 was enriched in ATPase, ion transmembrane transport GO, and NOD or TNFα pathways (Figure 1C; Figure S3). C1 is characterized by pathways and processes related to chemokines, pathogen immune defence, calcium, and GPCR signalling. C2 has no process or pathway annotated, probably due to low differential mRNA expression associated. C3 expresses nucleoside triphosphate and tuberculosis responses (Figure S3). C4 and C1 share leukocyte transendothelial migration, granule secretion, oxidative stress, and immune response mechanisms like TLR/NOD (Figure 1C; Figure S3). Besides this, C4 also is enriched in processes and pathways related metabolic and cellular activity (Figure 1C; Figure S3). Finally, C5 presents immune-related mechanisms. TTRUST analysis revealed that C1 was enriched in eosinophil transcription factors, C3 in interferon responses, and C5 in NF-κB and STAT-6 (Figure S3). C4 had no specific transcription factor network, whereas C1 presents highest number of associated transcription factors, which could describe eosinophils in different maturation state. This is also supported with the data showing that both C1 and C4 share pathways and functions related to immunity (against pathogens, granule secretion, oxidative stress) but C4 presents higher amount of asthma related gene expression and increase cellular metabolism and activation processes, a sign of cellular activation. According to our results, blood eosinophil transcriptomics is homogeneous, compromising the existence of intrinsic subpopulations. A 2016 study using mouse asthma models identified two different eosinophil lung subpopulations (resident and inflammatory) distinguished by phenotype and function as well as their possible reaction to IL-5, which opened the field to the idea of the existence of eosinophil subpopulations.3 The independency of eosinophils form IL-5 was recently questioned, mice-anti-IL-5 treatment depleted all eosinophil populations, suggesting that eosinophils exist on an activation continuum.4 As previously mentioned, other studies also proposed different eosinophil subtypes,1 most of them described in mouse, such as eosinophils with differential expression of GR-1 and cytokine expression in lung in allergic reactions.5 Many studies on mice intestinal diseases have also shown different eosinophil subpopulations, such as CD11chi intestinal antigen presenting eosinophils that differ from those from the intestine lamina propria and blood.6 A study from Diny and coworkers shed light on the eosinophil differences in intestine describing that the aryl hydrocarbon receptor partially shapes eosinophil transcriptome in response to environmental triggers.7 Furthermore, scRNAseq and in vivo experiments of gastrointestinal disease mice models concluded that eosinophil sub specialization is a continuous differentiation process controlled by the tissue milieu, its signalling molecules and microbiota, a process that is sustained on their lineage plasticity and the sequential ontogeny observed for the eosinophil precursor-blood-intestine subpopulations in the trajectory analysis.8 In accordance, Abdala–Valencia et al also proposed that it is the tissue microenvironment which shapes the eosinophils phenotype and function.9 These data support our findings, with all clusters having immune response pathways and eosinophil activation being prominent in C4 (increased S100A4, S100A8, CLC, SELL, associated with worst lung function or male sex), which reinforce that there might not be intrinsic eosinophil subpopulations in blood, but rather when the cell arrives to the tissue, then microenvironment changes eosinophils by virtue of their plasticity. Recently it has been published that CD62Llow inflammatory blood eosinophils are abundant in asthmatics.10 Conversely, we found that C4 does not fit this paradigm of CD62Llow inflammatory eosinophils, as it was increased in the asthmatic with worst lung function and presents higher Th2 asthma score, in addition to the fact that it is the most distinct cluster between healthy volunteers and asthmatics. A possibility is that being CD62L cytokine-dependent (downregulated by IL-5 and GM-CSF and upregulated by IFNγ),11 its levels on eosinophils may be affected by tissue microenvironment and disease status more than define eosinophil subpopulations. Being this study the first report using scRNAseq of human blood eosinophils we provide with an unsupervised approach into how are eosinophils at a transcriptomic level, proving that there are indeed differences between the clusters, but the overall results highlight certain cellular homogeneity in blood and differences attributable to activation states, differing from results for other immune cells like lymphocytes or monocytes,12 and from eosinophils located at tissue.8 Furthermore, as all clusters homogeneously represented in healthy and asthmatics blood eosinophils points to the hypothesis that eosinophils have high plasticity and acquire differential maturation, phenotype and functional states after tissue localization and stimulation as previously mentioned. It is worth mentioning that this study also presents limitations, first, although the number of sequenced cells is fine (loaded 20,000–40,000 cells per sample, retaining around 23,031 sequenced cells after filtering, a high number given eosinophil fragility) the sample size is small, and accounts for a small representation of the asthmatic population heterogeneity, which should be improved in future studies, where the inclusion of more and different subjects of study could provide even with data on asthma phenotypes and endotypes and their relationship with blood eosinophils. Moreover, all patients included in the study were phenotyped as severe eosinophilic asthmatics (and over time were treated with biologics), but present heterogeneous characteristics (lung function, FeNO, exacerbations…), which can be observed for C4, that mainly derives from one subject with worst lung function, high FeNO and no exacerbations the last year, proving that, as previously mentioned, more studies should be performed in order to better define eosinophils in the context of asthma heterogeneity. In conclusion, this is the first human blood eosinophil scRNAseq from healthy and asthmatic showing that at the transcriptional level, eosinophils are a homogeneous population (even between healthy/asthmatic conditions) with few gene and in silico differences resembling transient cellular activation phenotypes modulated by homeostatic and pathophysiological status. VdP conceived of the manuscript and designed the study. JMR-M, SN-G, SC, AB, CL-S, MG-M, ZG-dC, and JAC performed experiments; JMR-M, SN-G, AD, CR-R, CT, FS-C, and VdP performed data analysis. D-B, JB, MV-M, EJP-R, OS-P, MJR-N, and JS included patients and retrieved clinical data. JMR-M, SN-G, and VdP edited and wrote the manuscript. All authors contributed to the article and approved the submitted version. The authors would like to thank all the patients for their voluntary participation as well as all technical and nursing staff involved in the project (Manuela Garcia del Potro, Erica Aguado Wakui, Esther Gamella Álvarez, María Remedios Marquina Valero, Almudena Batanero Rodríguez and Raquel García Latorre). The authors also recognize Oliver Shaw, for his revision and editing in English. This work was supported by ISCIII—Instituto de Salud Carlos III and co-funded by the European Union, FIS (Fondo de Investigación Sanitaria—Spanish Health Research Fund) grants PI21/00896 and FI19/00067; Miguel Servet Program (CP23/00017); Ciber de Enfermedades Respiratorias (CIBERES); Ayudas para el fomento de la investigación de la Fundación de la SEAIC grants 22A07 and A21_09; Comunidad de Madrid grant PEJ-2021-AI_BMD-22320 and FEDER funds (Fondo Europeo de Desarrollo Regional). JMR-M reports receiving payments for lectures and educational events form Astra Zeneca and GSK. D-B reports having been under contract with the Instituto de Salud Carlos III (Rio Hortega Research Contract). MJR-N reports receiving research grant support from AstraZeneca, and having received payments for lectures from AstraZeneca and SANOFI. MV-M reports receiving payments for lectures and support for attending meetings and/or travel by AstraZeneca, GSK, Gebro, and Organon S.A.; as well as having received grant support for research from Organon S.A. VdP has received honoraria (advisory board, speaker) and/or institutional grant/research support from AstraZeneca and GSK and has held an unpaid leadership or fiduciary role in committees belonging to the EAACI. The rest of authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author, [VdP], upon reasonable request, and scRNAseq is deposited in the repository: https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14010. Figure S1. Figure S2. Figure S3. Data S1. Table S1. Table S2. Table S3. Table S4. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

SUMMARY:Spatial transcriptomics has changed our way to study tissue structure and cellular organization. However, there are still limitations in its resolution, and most available platforms do not reach a single cell resolution. To address this issue, we introduce SpatialDDLS, a fast neural network-based algorithm for cell type deconvolution of spatial transcriptomics data. SpatialDDLS leverages single-cell RNA sequencing data to simulate mixed transcriptional profiles with predefined cellular composition, which are subsequently used to train a fully connected neural network to uncover cell type diversity within each spot. By comparing it with two state-of-the-art spatial deconvolution methods, we demonstrate that SpatialDDLS is an accurate and fast alternative to the available state-of-the art tools.AVAILABILITY AND IMPLEMENTATION:The R package SpatialDDLS is available via CRAN-The Comprehensive R Archive Network: https://CRAN.R-project.org/package=SpatialDDLS. A detailed manual of the main functionalities implemented in the package can be found at https://diegommcc.github.io/SpatialDDLS.

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and, therefore, do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here we present the iFlpMosaics toolkit comprising a large set of new genetic tools and mouse lines that enable recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wild-type and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by fluorescence-activated flow cytometry and single-cell RNA sequencing. iFlpMosaics facilitate the induction and analysis of genetic mosaics in any quiescent or progenitor cell, and for any given single or combination of floxed genes, thus enabling a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis and disease.

Extensive genetic studies have elucidated cardiomyocyte differentiation and associated gene networks using single-cell RNA-seq, yet the intricate transcriptional mechanisms governing cardiac conduction system (CCS) development and working cardiomyocyte differentiation remain largely unexplored. Here we show that mice deleted for Dhx36 (encoding the Dhx36 helicase) in the embryonic or neonatal heart develop overt dilated cardiomyopathy, surface ECG alterations related to cardiac impulse propagation, and (in the embryonic heart) a lack of a ventricular conduction system (VCS). Heart snRNA-seq and snATAC-seq reveal the role of Dhx36 in CCS development and in the differentiation of working cardiomyocytes. Dhx36 deficiency directly influences cardiomyocyte gene networks by disrupting the resolution of promoter G-quadruplexes in key cardiac genes, impacting cardiomyocyte differentiation and CCS morphogenesis, and ultimately leading to dilated cardiomyopathy and atrioventricular block. These findings further identify crucial genes and pathways that regulate the development and function of the VCS/Purkinje fiber (PF) network.