ABSTRACT Single-cell sequencing has advanced the study of cell-cell communication, yet most methods focus on intercellular ligand-receptor interactions while neglecting downstream intracellular signalling cascades and the possibility that downstream target genes themselves encode ligands, thereby propagating communication across multiple cells. We present IntraTalker+CrossTalkeR that combines intracellular (IntraTalker) and intercellular (CrossTalkeR) signalling from multimodal single-cell data. IntraTalker infers cell-type-specific transcription factor activities and constructs receptomes that link receptors to downstream target genes, which are then integrated with ligand-receptor predictions in CrossTalkeR. To prioritize signalling receptors, the framework performs in silico receptor perturbation. In a murine bone marrow dataset, this recovered the known function of the Il1r1 receptor in driving myeloid progenitor cell-state changes. In a human myocardial infarction dataset, it predicted a novel role for IGF1R signalling in driving the differentiation of fibroblasts towards progenitor fibroblast states.

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis-inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12-abundant reticular (CAR) progenitor cells, resulting in loss of their hematopoiesis-support and upregulation of osteogenic and pro-apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury-specific, Wnt-dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal β-catenin expression is linked to fibrosis in patients, and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

MOTIVATION:Combining single-cell sequencing with ligand-receptor (LR) analysis paves the way for the characterization of cell communication events in complex tissues. In particular, directed weighted graphs naturally represent cell-cell communication events. However, current computational methods cannot yet analyze sample-specific cell-cell communication events, as measured in single-cell data produced in large patient cohorts. Cohort-based cell-cell communication analysis presents many challenges, such as the nonlinear nature of cell-cell communication and the high variability given by the patient-specific single-cell RNAseq datasets. RESULTS:Here, we present scACCorDiON (single-cell Analysis of Cell-Cell Communication in Disease clusters using Optimal transport in Directed Networks), an optimal transport algorithm exploring node distances on the Markov Chain as the ground metric between directed weighted graphs. Benchmarking indicates that scACCorDiON performs a better clustering of samples according to their disease status than competing methods that use undirected graphs. We provide a case study of pancreas adenocarcinoma, where scACCorDion detects a sub-cluster of disease samples associated with changes in the tumor microenvironment. Our study case corroborates that clusters provide a robust and explainable representation of cell-cell communication events and that the expression of detected LR pairs is predictive of pancreatic cancer survival. AVAILABILITY AND IMPLEMENTATION:The code of scACCorDiON is available at https://scaccordion.readthedocs.io/en/latest/. and https://doi.org/10.5281/zenodo.15267648. The survival analysis package can be found at https://github.com/CostaLab/scACCorDiON.su.

Computational trajectory analysis is a key computational task for inferring differentiation trees from this single-cell data. An open challenge is the prediction of complex and multi-branching trees from multimodal data. To address these challenges, we present PHLOWER (decomposition of the Hodge Laplacian for inferring trajectories from flows of cell differentiation), which leverages the harmonic component of the Hodge decomposition on simplicial complexes to infer trajectory embeddings from single-cell multimodal data. These natural representations of cell differentiation facilitate the estimation of their underlying differentiation trees. We evaluate PHLOWER through benchmarking with multi-branching differentiation trees and using kidney organoid multimodal and spatial single-cell data. These demonstrate the power of PHLOWER in both the inference of complex trees and the identification of transcription factors regulating off-target cells in kidney organoids. Thus, PHLOWER enables inference of complex branching trajectories and prediction of transcriptional regulators by leveraging multimodal data.

Cellular differentiation, the process by which a cell changes its chromatin and expression programs to acquire more specialized functions, is not only crucial in the development of multicellular organisms but also key during onset and progression of diseases. Transcription factors, which are proteins binding to regulatory DNA regions (open chromatin regions), are key regulators of gene expression thereby orchestrating cellular differentiation processes. Dissecting these key regulatory events will help to develop protocols for cellular reprogramming or ex vivo differentiation in e.g. organoids and to understand disease-related differentiation processes for potential therapeutic interventions. In this context, a unique resource to understand the interplay between chromatin, regulatory signals (transcription factor binding), and expression changes during cellular differentiation, is multi-modal single-cell sequencing, which can measure both full expression programs and genome-wide open chromatin. However, the associated experimental protocols dissociate cells, making it impossible to track how an individual cell differentiates over time experimentally. ### Competing Interest Statement The authors have declared no competing interest.

Recent studies highlight cellular crosstalk between fibrosis-inducing hematopoietic cells and fibrosis-driving stromal cells in bone marrow (BM) niche transformation in myeloproliferative neoplasms (MPNs). Absence of CXCL4 (PF4) reduces stromal cell activation, BM fibrosis and profibrotic pathways in megakaryocytes (MKs). Understanding this crosstalk is crucial for therapeutic targeting, but challenging as signaling pathways dependent on direct physical interactions are lost in current single-cell sequencing protocols.We aimed to dissect physical interactions between PF4-expressing cells (megakaryocytes, macrophages, monocytes) and Gli1+ stromal cells. Genetic fate tracing and BM transplantation from Pf4-ZsGreen mice into Gli1CreERt2;tdTomato mice tracked cell fate post-transduction with TPO vector inducing BM fibrosis. As mice showed fibrosis symptoms, Gli1+ cells and Pf4+ cells were sorted individually (singlets) and as multiplets (tdTomato+ ZsGreen+).We reveal an unprecedented representation of monocytes, macrophages, CAR stromal cells and endothelial cells in BM fibrosis with 27,000 singlets and 35,927 physically interacting multiplets. Computational analysis of singlets and multiplets identified changes in physically interacting cells and underlying cellular communication networks in disease. Computational analysis and imaging reveal various interacting niches that adapt in disease, and a macrophage subtype that acts as an "interaction hub," upregulating Spp1 expression in macrophages and osteoCAR cells. By directly targeting this interaction using an Spp1 genetic knockout in HSCs, we show an amelioration in BM fibrosis and a reduced anemic phenotype, hallmarks of advanced MPN. This novel approach allows us to decipher intricate cellular interactions in BM fibrosis pathogenesis and identify novel therapeutic targets that directly impact BM fibrosis.

Postnatal establishment of enteric metabolic, host-microbial and immune homeostasis is the result of precisely timed and tightly regulated developmental and adaptive processes. Here, we show that infection with the invasive enteropathogen Salmonella Typhimurium results in accelerated maturation of the neonatal epithelium with premature appearance of antimicrobial, metabolic, developmental, and regenerative features of the adult tissue. Using conditional Myd88- deficient mice, we identify the critical contribution of immune cell- derived mediators. Cytokine stimulation of neonatal intestinal epithelial stem cell organoids suggests a network of synergistic and antagonistic cytokine effects with a significant contribution of IL- 22, IL- 4/IL-13, TNF, and IL- 6 to infection- induced enterocyte reprogramming. Our findings demonstrate that the infection- associated immune cell activation disrupts physiological postnatal tissue maturation and may thereby worsen clinical outcomes and alter the neonatal- adult transition.

Comparing graphs by means of optimal transport has recently gained significant attention, as the distances induced by optimal transport provide both a principled metric between graphs as well as an interpretable description of the associated changes between graphs in terms of a transport plan. As the lack of symmetry introduces challenges in the typically considered formulations, optimal transport distances for graphs have mostly been developed for undirected graphs. Here, we propose two distance measures to compare directed graphs based on variants of optimal transport: (i) an earth movers distance (Wasserstein) and (ii) a Gromov-Wasserstein (GW) distance. We evaluate these two distances and discuss their relative performance for both simulated graph data and real-world directed cell-cell communication graphs, inferred from single-cell RNA-seq data.

Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroy-ing tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-re-ceptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in pa-tients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

Datasets for PILOT Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we are still lacking computational methods for the analysis of single-cell and pathomics data at a patient level for finding patient trajectories associated with diseases. This is challenging as a single-cell/pathomics data is represented by clusters of cells/structures, which cannot be compared with other samples. We propose here patient Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two single single-cell experiments. This allows us to perform unsupervised analysis at the sample level and to uncover trajectories associated with disease progression. Moreover, PILOT provides a statistical approach to delineate non-linear changes in cell populations, gene expression and tissues structures related to the disease trajectories. We evaluate PILOT and competing approaches in disease single-cell genomics and pathomics studies with up to 1.000 patients/donors and millions of cells or structures. Results demonstrate that PILOT detects disease-associated samples, cells, and genes from large and complex single-cell and pathomics data.

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that arises from clonal proliferation of hematopoietic stem cells (HSCs) and leads to progressive bone marrow (BM) fibrosis, resulting in extramedullary hematopoiesis (typically in the spleen), BM failure, and ultimately death. Using single-cell RNAseq, we showed disease-specific upregulation of the alarmin complex S100A8/S100A9 in fibrosis-driving mesenchymal stromal cell (MSC) populations (Leimkühler et al., 2021). Importantly, targeting these alarmins with the small molecular oral inhibitor tasquinimod stopped the progression of fibrosis in murine JAK2V617F PMF models. Aims: The questions remained whether the effect of alarmin inhibition of Tasquinimod is more relevant in hematopoietic or stromal cells and how Tasquinimod affects the hematopoiesis-stromal cell crosstalk. Methods: Here, we analyzed the effect of Tasquinimod on hematopoietic cells and stromal cells using RNA sequencing in a JAK2V617F murine PMF model, systematically analyzed the effect of stromal versus hematopoietic S100A9 inhibition using a genetic knockout model, and determined the consequences of S100A9 overexpression using a genetic knock-in model and lentiviral overexpression. Results: Receptor-ligand analysis of RNA sequencing data demonstrated that Tasquinimod indeed targets the disease-specific alarmin-driven interaction between CD41+ megakaryocytes, Lin-Sca1+ stromal cells and CD11b+Gr1- monocytes. The increased alarmin signalling originated from JAK2V617F malignant hematopoietic cells and communicated to stromal cells. In addition, TGFb-based interactions between CD41+ megakaryocytes and Lin-Sca1+ stromal cells were significantly reduced, which shows a direct anti-fibrotic effect of Tasquinimod as TGFb is supposed to be the master switch of fibrosis. Additionally, we observed a significant decrease in PI3K and Myc pro-proliferative signalling in the JAK2V617F mutant hematopoietic cells. In line with this, we functionally demonstrated that tasquinimod specifically induces apoptosis in JAK2V617F cells. We thus concluded that increased alarmin signalling originates from JAK2V617F malignant hematopoietic cells and communicates to stromal cells leading to initiation of fibrosis. We show that only MSCs in co-culture with malignant JAK2V617F cells upregulate alarmins and TGFb and decrease CXCL12 expression, gaining an overall pro-inflammatory and pro-fibrotic phenotype, combined with a loss of hematopoietic support. In line with this conclusion, we confirmed that genetic knockout of S100A8/S100A9 in the hematopoietic compartment, but not in stromal cells, alone ameliorates myelofibrosis, reduces splenomegaly and myeloproliferation in both ThPO- and JAK2V617F-induced PMF. Summary/Conclusion: We demonstrate that alarmins need to be secreted and that transcriptional overexpression by lentiviral overexpression and genetic hematopoietic knock-in of S100A8/S100A9 do not lead to myeloproliferation or to an MPN phenotype as S100A8/S100A9 levels do not increase. The S100A8/S100A9 heterodimer levels determined by ELISA correlate with the MPN severity in murine models but also in large cohorts of patient samples. As a next step, we will answer whether alarmins can act not only as a biomarker for progression but also for tasquinimod response in a clinical trial (tasqForce MPN), which will be started by the end of the year (PIs: P. te Boekhorst, M. Crysandt, Scientific PI: R. Schneider). In murine models, S100A8 levels in the plasma correlate with the response to Tasquinimod making S100A8/S100A9 a putative actionable biomarker. Keywords: Bone Marrow Fibrosis, Bone marrow microenvironment, Myelofibrosis

Cardiac remodeling occurs frequently in chronic kidney disease patients and affects quality of life and survival. Current treatment options are highly inadequate. As kidney function declines, numerous metabolic pathways are disturbed. Kidney and heart functions are highly connected by organ crosstalk. Among others, altered volume and pressure status, ischemia, accelerated atherosclerosis and arteriosclerosis, disturbed mineral metabolism, renal anemia, activation of the renin-angiotensin system, uremic toxins, oxidative stress and upregulation of cytokines stress the sensitive interplay between different cardiac cell types. The fatal consequences are left-ventricular hypertrophy, fibrosis and capillary rarefaction, which lead to systolic and/or diastolic left-ventricular failure. Furthermore, fibrosis triggers electric instability and sudden cardiac death. This review focuses on established and potential pathophysiological cardiorenal crosstalk mechanisms that drive uremia-induced senescence and disease progression, including potential known targets and animal models that might help us to better understand the disease and to identify novel therapeutics.

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-β-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-β-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage.

The increasing availability of single-cell multi-omics data allow to quantitatively characterize gene regulation. We here describe scMEGA (Single-cell Multiomic Enhancer-based Gene Regulatory Network Inference) to infer gene regulatory network by combining single cell gene expression and chromatin accessibility profiles. This allows to study complex gene regulation mechanisms for dynamic biological processes, such as cellular differentiation and disease development. We provide a case study on gene regulatory networks controlling myofibroblast activation in human myocardial infarction.

The goal of this study is to elucidate the cellular origin and underlying mechanisms of cells contributing to neointima formation following stent placement.

Bone remodeling occurs actively alongside bone marrow (BM) fibrosis in the context of myeloproliferative neoplasms (MPN). Osteosclerosis in MPN is characterized by para-trabecular apposition of new bone and increase in bone density. Yet, the pathogenesis of osteosclerosis is largely unknown and it remains an unanswered question how the 1) peri-vascular, and 2) endosteal stromal niche contribute to the formation of new bone. Here, we employed genetic fate tracing and high-resolution imaging combined with single-cell RNA sequencing (scRNAseq) to map out the BM stromal niche in steady-state, after transplant of control or thrombopoietin (ThPO)-overexpressing hematopoietic stem and progenitor cells (HSPCs) which robustly and with defined kinetics results in BM fibrosis/osteosclerosis. High-resolution confocal imaging revealed that PdgfrbCreERt2-derived tdTomato+ cells are abundant throughout the whole marrow, growth plate and bone. Gli1CreERt2;tdTomato+ have a distinct localization specifically at the growth plate, in the trabecular region of the metaphysis and around the central artery in the diaphysis. Grem1CreERt2;tdTomato+ cells are the least abundant, specifically located at the growth plate. Integrated data sets provided a high resolution of the BM stroma representing Cxcl12-abundant reticular (CAR) cells, fibroblasts (FBs), osteo-lineage cells (OLCs), osteoblasts (OBs), (pre)chondrocytes, Schwann cells and endothelial subpopulations. We next asked how these different subsets contribute to BM fibrosis and osteosclerosis. CAR cells were functionally reprogrammed in fibrosis: they lost their hematopoiesis-supporting capacity, gained a pro-fibrotic phenotype, produced extra-cellular matrix (ECM) and were less frequent compared to control conditions. BM-resident FBs rather gained a pro-inflammatory phenotype, comparable to inflammatory fibroblasts described in solid organ fibrosis, and were enriched in fibrosis but did not show a "pro-fibrotic" expression profile. Strikingly, the major expansion of tdTomato+ stromal cells occurred at the metaphysis, overlapping with increased reticulin deposition in this region. Deconvolution revealed that mainly OLCs, pre-chondrocytes and chondrocytes but also FBs reside in the metaphysis. Cell trajectory analysis suggested that a subset of OLCs acts as a mesenchymal precursor reservoir for OBs, chondrocytes and FBs. In line with our hypothesis, only metaphyseal, but not diaphyseal, PdgfrbCreERt2;tdTomato+ cells from steady-state mice harbored CFU-F capacity and gave rise to colonies in vitro. Receptor-ligand interaction demonstrated that OLCs function as important information-hubs to CAR cells after BM transplantation compared to steady-state bone. This pro-regenerative phase is characterized by upregulation of adipogenesis-related genes, potentially prompting the BM niche to recover hematopoiesis. In response to a fibrotic cue, OLCs expand, lose cellular crosstalk reflected by decreased receptor-ligand pairs, downregulate adipogenic signatures but upregulate osteogenic signatures, thus skewing the BM stroma towards osteogenesis. Time-course imaging of the stepwise invasion of Gli1CreERt2;tdTomato+ cells into the BM from the growth plate additionally highlighted the active bone remodeling occurring at the chondrocyte-OLC border during fibrosis. µCT imaging revealed increased ossification specifically at the metaphyseal region. Pathways analysis demonstrated that metaphyseal cells remain active even in progressed fibrosis and show enriched Wnt, PI3K and ECM receptor signaling. Interestingly, Wnt pathway inhibitors were downregulated in pro-fibrotic-CAR cells, whereas Wnt signaling was increased in OLCs as mesenchymal progenitor cells upon fibrotic transformation. In summary, we provide evidence that active bone remodeling is co-occurring with the fibrotic transformation with a skewing of stromal-cell fate towards osteogenesis rather than adipogenesis. Our analysis highlights the functional differences of metaphyseal and diaphyseal macro-niches within bone and postulates that a metaphyseal stromal progenitor is activated in an injury-specific manner, being an attractive cellular target.