Biology subjects

Krammer, T.

Publications and source records attributed to Krammer, T..

11 recordsLinked to original sources

bioRxiv2026

TREM2 drives accumulation of pro-scarring monocyte-derived macrophages in the infarcted myocardium

Myocardial infarction is a leading cause of death and disability worldwide. Ischemic injury leads to irreversible loss of cardiomyocytes, the contractile cells of the heart, and formation of a fibrotic scar. After infarction, macrophages massively infiltrate the heart and orchestrate the tissue repair process by removing dead cells and modulating fibroblast activation for scar formation. We previously demonstrated that diverse monocyte-derived macrophage populations dynamically accumulate in the heart following myocardial infarction, notably a pro-repair Trem2hi subset. In this study, we leveraged spatial transcriptomics, single-cell RNA-seq, and functional assays to elucidate the role of TREM2 in driving macrophage-mediated cardiac tissue repair post-infarction. We show that Trem2hi macrophages localize in scarring areas of the infarcted myocardium in the vicinity of collagen-producing myofibroblasts. In Trem2-/- mice, cardiac accumulation of monocyte-derived macrophages with a pro-scarring matrisome-associated macrophage signature was reduced. TREM2 deficiency was functionally associated with reduced fibroblast proliferation, accumulation of myofibroblasts, decreased collagen deposition in the infarcted heart, and increased infarct size. In vitro, we show that TREM2 mediates efferocytosis-induced pro-fibrotic gene expression and promotes macrophage ability to induce fibroblast migration. IL-4 priming of bone marrow-derived macrophages further increased the pro-fibrotic response in macrophages, suggesting that IL-4 and efferocytosis act synergistically to drive this phenotype. Altogether, our results show that TREM2 is essential for the accumulation and function of pro-scarring monocyte-derived macrophages in the infarcted myocardium.

immunology↗

bioRxiv2026

Minimal essential requirements for neural tube self-organisation

The reliable generation of diverse cell types in precise proportions is essential for the formation of functional tissues during embryonic development. Three-dimensional organoid models derived from pluripotent stem cells (PSCs) provide powerful systems for identifying principles governing tissue self-organisation. Neural tube organoids (NTOs), initiated from single PSCs with a pulse of retinoic acid (RA), self-organise into structures containing floorplate cells that secrete SHH morphogen to pattern adjacent neural tissue. Yet, how the initial cellular diversity arises and how appropriate cell-type proportions are allocated has remained unclear. Here, using time-resolved single-cell transcriptomics, quantitative immunofluorescence, and dynamical systems modelling we show that RA triggers a transient co-expression state of the transcription factors PAX6 and FOXA2 from which cells asynchronously resolve into two opposing fates: PAX6 neural precursors and FOXA2 floorplate precursors. PAX6 and FOXA2 are both necessary and sufficient to reconstitute self-organisation, establishing these transcription factors as key determinants of emergent tissue pattern. Rather than operating as a simple feed-forward system in which cells are guided solely by RA, feedback between the alternative cell fates, mediated by BMP signalling from floorplate precursors, determines cell type proportions and ensures reproducible cell-type diversity in each NTO. This dual expression state was also identified in mouse embryos, demonstrating how in vitro models inform in vivo biology. These findings establish a general design strategy - symmetry breaking through opposing fate determinants coupled to proportioning via signal feedback control - that may operate broadly across developmental contexts to generate tissues with predictable cellular compositions.

developmental biology↗

bioRxiv2026

Neutrophil terminal programming in the ischemic heart drives fibrosis after myocardial infarction

Following myocardial infarction (MI), the heart undergoes massive neutrophil infiltration characterized by the emergence of distinct subsets, notably a SiglecF+ population that accumulates according to specific temporal dynamics. The mechanisms governing cardiac neutrophil heterogeneity and the subsequent functional impact of this diversity on tissue repair following myocardial infarction remain to be elucidated. Using single-cell RNA-sequencing of neutrophils in the heart and peripheral organs of infarcted mice, we here show that while acquisition of the SiglecF+ state only fully occurs in the ischemic heart tissue, MI primes neutrophils in the periphery to acquire Siglecf and to upregulate receptors for TGF{beta} and GM-CSF that drive acquisition of the SiglecF+ state. Ly6G targeting in vivo shifted cardiac neutrophils towards the SiglecF+ state at day 3 post-MI, induced the emergence of reprogrammed SiglecF+Ly6Glo and Retnlghi neutrophil states at day 5, and was associated with increased fibrosis of the infarct border zone. Mechanistically, Ly6G targeting reshaped the cardiac immune landscape with increased recruitment of pro-fibrotic {gamma}{delta} T cells and monocytes, and SiglecF+ neutrophils exerted direct pro-fibrotic effects on fibroblasts in a co-culture system. Altogether, our results indicate that peripheral neutrophil priming combined with their terminal programming towards a SiglecF+ state in the ischemic heart drives cardiac fibrosis after MI.

physiology↗

bioRxiv2025

Murine CMV Infection Unmasks Macrophage-Driven Inflammatory Cardiomyopathy in Pkp2, but not in Ttn Mutant Mice

2.BackgroundGenetic cardiomyopathies display variable penetrance and phenotypic expression, highlighting the influence of environmental modulators. Myocarditis, commonly triggered by cardiotropic viruses, overlaps clinically with genetic cardiomyopathies. Consequently, these infections are implicated as secondary factors that accelerate disease onset and progression, yet their precise impact in specific genetic settings remains unexplored. MethodsTo interrogate this, genetic mouse models heterozygous for a mutant allele of desmosomal plakophilin-2 (Pkp2+/-) or sarcomeric titin (Ttn+/-), genes frequently linked to acute myocarditis, were challenged with murine cytomegalovirus (MCMV) to determine how latent infection influences myocardial inflammation, tissue remodeling, and cardiac performance. Integrated experimental approaches, including echocardiography, histology, flow cytometry, single-cell RNA sequencing, as well as cytokine and kinome analyses, defined immune and signaling responses in infected versus non-infected hearts. ResultsAcute, MCMV-induced viral myocarditis and subsequent latent MCMV infection unmasked early disease onset in Pkp2+/- animals, leading to progressive systolic impairment, whereas in Ttn+/- mice cardiac structure and function remained preserved throughout infection. Cardiac immune profiling uncovered infection- and genotype-specific divergence: both genetic models showed a stable myocardial effector-memory CD8+ T-cell response to MCMV, but only Pkp2+/- hearts recruited additional Ly6C+ CCR2+ monocytes and macrophages with distinct inflammatory signatures. In the absence of infection, Pkp2 insufficiency initiated subclinical CCL2 secretion and subsequent recruitment of CCR2+ cells, reflecting early immune activation preceding age-associated functional and structural decline. At this stage, cytokine and kinase evaluations indicated a balance between proinflammatory and compensatory signals. However, with aging or following MCMV challenge, this balance shifted towards persistent inflammation, evidenced by chronic upregulation of cytokines and activation of signaling pathways, which ultimately led to adverse effects and myocardial dysfunction. ConclusionsManifestation of genetic cardiomyopathies depends on interactions between inherited susceptibility and environmental stressors. Here, we show that cytomegalovirus infection intensifies inflammation in PKP2-related cardiomyopathy. In contrast, TTN-linked cardiomyopathy does not exhibit increased inflammation under the same conditions. For individuals carrying desmosomal variants, infection control and tailored anti-inflammatory strategies may attenuate or delay disease manifestation and progression.

immunology↗

bioRxiv2025

CD4+ T cells fuel the Duchenne cardiomyopathy

Duchenne muscular dystrophy (DMD) is a X-linked genetic disorder, in which cardiomyopathy represents a major cause of mortality. Although myocardial inflammation and fibrosis are hallmarks of the disease, the role of adaptive immunity in cardiac pathology remains poorly defined. Using the Mdx mouse model, we demonstrate that T and B lymphocytes accumulate in the heart and that activation occurs in heart-draining lymph nodes at an early disease stage. Mdx mice lacking adaptive immunity (Mdx-Scid) were protected from myocardial fibrosis and hypertrophic remodeling. Single-cell RNA sequencing revealed expansion of an inflammatory, matrisome-associated macrophage subset in Mdx but not Mdx-Scid hearts. Genetic deficiency or depletion of CD4+ T cells reduced left ventricular fibrosis and preserved systolic function. Moreover, adoptive transfer of T cells von Mdx mice induced myocardial fibrosis and dysfunction in healthy recipients. Our results identify autoreactive CD4+ T cells as key drivers of DMD-associated cardiomyopathy and suggest targeted modulation of adaptive immune responses as a potential therapeutic approach in DMD.

immunology↗

bioRxiv2025

Targeting platelet GPVI or GPIIb/IIIa only minimally affect acute inflammation and cardiac repair in experimental permanent myocardial infarction

BackgroundBeyond their role in hemostasis, platelets are recognized as key regulators of inflammatory responses in ischemic diseases, including cardiac ischemia/reperfusion (I/R) injury with key roles of platelet membrane glycoproteins (GP)VI and IIb/IIIa. However, whether platelet-driven thrombo-inflammatory pathways affect acute inflammation and cardiac repair processes in permanent, non-reperfused myocardial infarction (MI) is unknown. MethodsWe targeted GPVI and GPIIb/IIIa in experimental permanent MI in mice. Cardiac, bone marrow, and blood innate immune responses were evaluated by flow cytometry and single-cell RNA-sequencing. Survival and cardiac repair were assessed over the inflammatory and scar formation phase, until day 10 after permanent MI. ResultsPlatelet GPVI immunodepletion by injection of the anti-GPVI antibody JAQ1 did not affect levels of neutrophil or monocyte subsets (Ly6Chi and Ly6Clow) in the bone marrow and blood, and did not alter accumulation of monocytes, macrophage subsets (defined by expression of MHCII and TIM4), or neutrophil subsets (SiglecFhi/low) in the infarcted heart on day 4. GPVI depletion only had a minimal effect on cardiac repair, slightly decreasing interstitial fibrosis in the infarct border zone on day 10. Four days after MI, GPIIb/IIIa inhibition by JON/A-F(ab')2 had no effect on cardiac or systemic innate immune cell levels as measured by flow cytometry and did not affect composition and transcriptomic profile of the cardiac immune infiltrate as revealed by single-cell RNA-sequencing. GPIIb/IIIa inhibition did not improve cardiac remodeling, and was even associated with an increased mortality rate over 10 days post-MI. ConclusionTargeting the GPVI-GPIIb/IIIa axis only had minor effects on post-MI inflammatory responses and cardiac wound healing in permanent myocardial ischemia. Our findings demonstrate that the therapeutic benefits of inhibiting platelet-driven thrombo-inflammation are particularly relevant in the subacute reperfusion phase.

physiology↗

bioRxiv2024

Identifying Modulators of Cellular Responses by Heterogeneity-sequencing

The response of individual cells to drug treatment, virus infections or other molecular stimuli is highly heterogeneous and depends on the cells initial state. Library preparation for single-cell transcriptomics is destructive, precluding a direct comparison between the initial state and the stimulus outcome. Consequently, current methods are restricted to identifying correlative associations rather than resolving causal drivers of heterogeneous outcomes. We developed Heterogeneity-seq, which combines single-cell RNA-seq with metabolic RNA labeling (scSLAM-seq) and double machine learning to overcome this limitation. By leveraging simultaneous measurements of unlabeled and labeled RNA in individual cells, Heterogeneity-seq uncovers the transition from pre-stimulated cell states to distinct stimulation outcomes across thousands of cells. These links enable the identification of factors that causally govern heterogeneous cellular responses. We used Heterogeneity-seq to identify both known and novel genes that drive responses to drug treatment, as well as pro- and antiviral host factors governing cytomegalovirus infection.

bioinformatics↗

bioRxiv2024

Neural tube organoid generation: a robust and reproducible protocol from single mouse embryonic stem cells

The development of mammals is a highly complex process, characterized by the necessity for precise concentration- and time-dependent signaling for correct pattern formation and morphogenesis. Despite considerable technological advancements and knowledge gathered, numerous aspects of mammalian development remain elusive. When examining the entire organism, it becomes challenging to disentangle the effects of individual pathways or the mechanism by which external stimuli guide the interference of surrounding tissues and factors. In addressing this complexity, three-dimensional (3D) in vitro models such as organoids have emerged as valuable tools. Organoids, derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), exhibit tissue-like features that closely resemble their in vivo counterparts in terms of expression patterns and functionality. Importantly, they offer accessibility for manipulation and extensive biological studies within a controlled experimental setting. Despite originating from pluripotent cultures, organoid systems often exhibit heterogeneity and substantial variability, limiting their utility when studying complex and intricate biological questions. Therefore, there is a pressing need for detailed protocols aimed at harmonizing procedures that result in high-quality reproducible data, reduction of materials used and which importantly permit the investigation of convoluted phenomena. In this context, we present an optimized protocol for the cultivation of neural tube organoids (NTOs) in vitro. By producing stable culture conditions and offering comprehensive troubleshooting strategies, this protocol enables the reliable and reproducible generation of NTOs which serve as an adequate model to study relevant scientific questions. SUMMARYThree-dimensional neural tube organoids (NTOs) derived from mouse embryonic stem cells are valuable tools to study the central nervous system during early development. Here, we present a step-by-step demonstration of an optimized protocol for cultivating NTOs in vitro, providing stable culture conditions and troubleshooting strategies for reliable and reproducible NTO generation.

developmental biology↗

bioRxiv2023

Clonal neural tube organoids self-organise floorplate through BMP-mediated cluster competition

The neural tube (NT) has been a hallmark example of embryonic induction and patterning whereby the notochord induces an organiser, the floorplate, that secretes Sonic Hedgehog (SHH) to pattern the surrounding field of neural progenitors. On the other hand, NT organoids (NTOs) formed from embryonic stem cells (ESCs) undergo spontaneous floorplate formation and patterning in the absence of their normal embryonic inducers. Understanding how stem cells undergo regulative organiser formation is a central challenge in biology. Here, we investigated the self-organisation of a SHH-expressing floorplate organiser using clonal NTOs. Expression of FOXA2, a floorplate transcription factor, was initially spatially scattered before resolving into multiple clusters. These FOXA2+ clusters underwent competition and physical sorting, resulting in a stable "winning" floorplate. We identified BMP signalling as a key governor of long-range cluster competition. FOXA2+ clusters expressed BMP4 ligand suppressing FOXA2 in receiving cells, while simultaneously expressing the BMP-inhibitor NOGGIN to secure FOXA2+ cluster survival. Genetic mutation of Noggin perturbed the floorplate not only in NTOs but also in vivo at the mid-hindbrain region of the mouse NT. These results demonstrate how the floorplate can form autonomously without its well-known inducer, the notochord, suggesting redundant mechanisms ensuring robustness. Defining molecular pathways that govern organiser self-organisation is critical in harnessing the developmental plasticity of stem cells toward directed tissue engineering.

developmental biology↗

bioRxiv2021

Spatial organisation and homeostasis of epithelial lineages at the gastroesophageal junction is regulated by the divergent Wnt mucosal microenvironment

The gastroesophageal junction (GEJ), where squamous and columnar epithelia meet, is a hotspot for Barretts metaplasia development, dysbiosis and carcinogenesis. However, the mechanisms regulating GEJ homeostasis remain unclear. Here, by employing organoids, bulk and single-cell transcriptomics, single-molecule RNA in situ hybridisations and lineage tracing, we identified the spatial organisation of the epithelial, stromal compartment and the regulators that maintain the normal GEJ homeostasis. During development, common KRT8 progenitors generate committed unilineage p63/KRT5-squamous and KRT8-columnar stem cells responsible for the regeneration of postnatal esophagus and gastric epithelium that meet at GEJ. A unique spatial distribution of Wnt regulators in the underlying stromal compartment of these stem cells creates diverging Wnt microenvironments at GEJ and supports their differential regeneration. Further, we show that these tissue-resident stem cells do not possess the plasticity to transdifferentiate to the other lineage with the altered Wnt signals. Our study provides invaluable insights into the fundamental process of GEJ homeostasis and is crucial for understanding disease development.

developmental biology↗

bioRxiv2019

scSLAM-seq reveals core features of transcription dynamics in single cells

Current single-cell RNA sequencing approaches gives a snapshot of a cellular phenotype but convey no information on the temporal dynamics of transcription. Moreover, the stochastic nature of transcription at molecular level is not recovered. Here, we present single-cell SLAM-seq (scSLAM-seq), which integrates metabolic RNA labeling, biochemical nucleoside conversion and single-cell RNA-seq to directly measure total transcript levels and transcriptional activity by differentiating newly synthesized from pre-existing RNA for thousands of genes per single cell. scSLAM-seq recovers the earliest virus-induced changes in cytomegalovirus infection and reveals a so far hidden phase of viral gene expression comprising promiscuous transcription of all kinetic classes. It depicts the stochastic nature of transcription and demonstrates extensive gene-specific differences. These range from stable transcription rates to on-off dynamics which coincide with gene-/promoter-intrinsic features (Tbp-TATA-box interactions and DNA methylation). Gene but not cell-specific features thus explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.

genomics↗